Homologous recombination (HR) is initiated by DNA double-strand breaks (DSB). However, it remains unclear whether single-strand lesions also initiate HR in genomic DNA. Chicken B lymphocytes diversify their Immunoglobulin (Ig) V genes through HR (Ig gene conversion) and non-templated hypermutation. Both types of Ig V diversification are initiated by AID-dependent abasic-site formation. Abasic sites stall replication, resulting in the formation of single-stranded gaps. These gaps can be filled by error-prone DNA polymerases, resulting in hypermutation. However, it is unclear whether these single-strand gaps can also initiate Ig gene conversion without being first converted to DSBs. The Mre11-Rad50-Nbs1 (MRN) complex, which produces 3′ single-strand overhangs, promotes the initiation of DSB-induced HR in yeast. We show that a DT40 line expressing only a truncated form of Nbs1 (Nbs1p70) exhibits defective HR-dependent DSB repair, and a significant reduction in the rate—though not the fidelity—of Ig gene conversion. Interestingly, this defective gene conversion was restored to wild type levels by overproduction of Escherichia coli SbcB, a 3′ to 5′ single-strand–specific exonuclease, without affecting DSB repair. Conversely, overexpression of chicken Exo1 increased the efficiency of DSB-induced gene-targeting more than 10-fold, with no effect on Ig gene conversion. These results suggest that Ig gene conversion may be initiated by single-strand gaps rather than by DSBs, and, like SbcB, the MRN complex in DT40 may convert AID-induced lesions into single-strand gaps suitable for triggering HR. In summary, Ig gene conversion and hypermutation may share a common substrate—single-stranded gaps. Genetic analysis of the two types of Ig V diversification in DT40 provides a unique opportunity to gain insight into the molecular mechanisms underlying the filling of gaps that arise as a consequence of replication blocks at abasic sites, by HR and error-prone polymerases.
An important class of chemotherapeutic drugs used in the treatment of cancer induces DNA damage that interferes with DNA replication. The resulting block to replication results in the formation of single-strand gaps in DNA. These gaps can be filled by specialized DNA polymerases, a process associated with the introduction of mutations or by recombination with an undamaged segment of DNA with an identical or similar sequence. Our work shows that diversification of the antibody genes in the chicken B cell line DT40, which is initiated by localized replication-stalling DNA damage, proceeds by formation of a single-strand intermediate. These gaps are generated by the action of a specific nuclease complex, comprising the Mre11, Rad50, and Nbs1 proteins, which have previously been implicated in the initiation of homologous recombination from double-strand breaks. However, in this context, their dysfunction can be reversed by the expression of a bacterial single-strand–specific nuclease, SbcB. Antibody diversification in DT40 thus provides an excellent model for studying the process of replication-stalling DNA damage and will allow a more detailed understanding of the mechanisms underlying gap repair and cellular tolerance of chemotherapeutic agents.
During meiosis, self-inflicted DNA double-strand breaks (DSBs) are created by the protein Spo11 and repaired by homologous recombination leading to gene conversions and crossovers. Crossover formation is vital for the segregation of homologous chromosomes during the first meiotic division and requires the RecA orthologue, Dmc1.We analyzed repair during meiosis of site-specific DSBs created by another nuclease, VMA1-derived endonuclease (VDE), in cells lacking Dmc1 strand-exchange protein. Turnover and resection of the VDE-DSBs was assessed in two different reporter cassettes that can repair using flanking direct repeat sequences, thereby obviating the need for a Dmc1-dependent DNA strand invasion step. Access of the single-strand binding complex replication protein A, which is normally used in all modes of DSB repair, was checked in chromatin immunoprecipitation experiments, using antibody against Rfa1. Repair of the VDE-DSBs was severely inhibited in dmc1Δ cells, a defect that was associated with a reduction in the long tract resection required to initiate single-strand annealing between the flanking repeat sequences. Mutants that either reduce Spo11-DSB formation or abolish resection at Spo11-DSBs rescued the repair block. We also found that a replication protein A component, Rfa1, does not accumulate to expected levels at unrepaired single-stranded DNA (ssDNA) in dmc1Δ cells. The requirement of Dmc1 for VDE-DSB repair using flanking repeats appears to be caused by the accumulation of large quantities of ssDNA that accumulate at Spo11-DSBs when Dmc1 is absent. We propose that these resected DSBs sequester both resection machinery and ssDNA binding proteins, which in wild-type cells would normally be recycled as Spo11-DSBs repair. The implication is that repair proteins are in limited supply, and this could reflect an underlying mechanism for regulating DSB repair in wild-type cells, providing protection from potentially harmful effects of overabundant repair proteins.
During meiosis, DNA is deliberately damaged by formation of double-strand breaks. Programmed breaks must be repaired for cell division to be completed. Break repair enables reciprocal exchange between parental chromosomes, and this exchange acts as a link between chromosomes before anaphase separation. These links are essential to ensure that maternal and paternal chromosomes segregate into different daughter cells. Meiosis has special mechanisms to ensure the repair creates sufficient reciprocal exchanges between parental chromosomes; Dmc1 protein is essential for these mechanisms to work. When Dmc1 is absent, programmed breaks accumulate with excess single-stranded DNA nearby. Using reporter constructs integrated into yeast, we examined repair of an experimentally induced break expected not to need Dmc1. When Dmc1 is absent, programmed breaks accumulate in single-stranded form, and the experimental break is not repaired. Either preventing formation of programmed breaks, or stopping DNA near them from becoming single-stranded, relieves this repair block. We conclude that repair proteins are likely to be in limited supply during meiosis, and they run out in cells lacking Dmc1 function. Limiting protein supply may be an important regulatory mechanism, protecting DNA from potentially damaging effects of oversupply.
For most organisms, chromosome segregation during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs) and repair of a subset of these DSBs as inter-homolog crossovers (COs). However, timing and levels of DSB formation must be tightly controlled to avoid jeopardizing genome integrity. Here we identify the DSB-2 protein, which is required for efficient DSB formation during C. elegans meiosis but is dispensable for later steps of meiotic recombination. DSB-2 localizes to chromatin during the time of DSB formation, and its disappearance coincides with a decline in RAD-51 foci marking early recombination intermediates and precedes appearance of COSA-1 foci marking CO-designated sites. These and other data suggest that DSB-2 and its paralog DSB-1 promote competence for DSB formation. Further, immunofluorescence analyses of wild-type gonads and various meiotic mutants reveal that association of DSB-2 with chromatin is coordinated with multiple distinct aspects of the meiotic program, including the phosphorylation state of nuclear envelope protein SUN-1 and dependence on RAD-50 to load the RAD-51 recombinase at DSB sites. Moreover, association of DSB-2 with chromatin is prolonged in mutants impaired for either DSB formation or formation of downstream CO intermediates. These and other data suggest that association of DSB-2 with chromatin is an indicator of competence for DSB formation, and that cells respond to a deficit of CO-competent recombination intermediates by prolonging the DSB-competent state. In the context of this model, we propose that formation of sufficient CO-competent intermediates engages a negative feedback response that leads to cessation of DSB formation as part of a major coordinated transition in meiotic prophase progression. The proposed negative feedback regulation of DSB formation simultaneously (1) ensures that sufficient DSBs are made to guarantee CO formation and (2) prevents excessive DSB levels that could have deleterious effects.
Formation of haploid gametes during meiosis relies on deliberate induction of DNA double-strand breaks (DSBs), followed by repair of a subset of DSBs as crossovers between homologous chromosomes. Crossovers form the basis of connections that enable homologs to segregate toward opposite spindle poles at meiosis I, thereby reducing ploidy. Thus, germ cells must generate enough DSBs to guarantee a crossover for every chromosome pair while avoiding an excessive number of DSBs that might endanger their genomes. Here, we provide insight into how this crucial balance is achieved. We identify C. elegans DSB-2 as a key regulator of DSB formation, and we propose that its association with chromatin is an indicator of DSB competence. Disappearance of DSB-2 is part of a coordinated transition affecting multiple distinct aspects of the meiotic program, and failure to form crossover-eligible recombination intermediates elicits a delay in DSB-2 removal and other transition events. Our data are consistent with a model in which meiotic DSB formation is governed by a negative feedback network wherein cells detect the presence of downstream crossover intermediates and respond by shutting down DSB formation, thereby ensuring that sufficient DSBs are made to guarantee crossovers while simultaneously minimizing the threat to genomic integrity.
DNA double-strand breaks (DSBs), which are formed by the Spo11 protein, initiate meiotic recombination. Previous DSB-mapping studies have used rad50S or sae2Δ mutants, which are defective in break processing, to accumulate Spo11-linked DSBs, and report large (≥ 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2Δ mutants. We therefore developed a novel method to map genome-wide, single-strand DNA (ssDNA)–associated DSBs that accumulate in processing-capable, repair-defective dmc1Δ and dmc1Δ rad51Δ mutants. DSBs were observed at known hot spots, but also in most previously identified “DSB-cold” regions, including near centromeres and telomeres. Although approximately 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1Δ shows that most of these regions have substantial DSB activity. Southern blot assays of DSBs in selected regions in dmc1Δ, rad50S, and wild-type cells confirm these findings. Thus, DSBs are distributed much more uniformly than was previously believed. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as a critical strand-exchange activity genome-wide, and confirm previous conclusions that Spo11-induced lesions initiate all meiotic recombination.
During meiosis, the two copies of each chromosome present in the full (diploid) genome come together and then separate, forming haploid gametes (sperm and eggs, in animals). Recombination, which swaps DNA between chromosomes, is critical for chromosome pairing and separation, and also promotes genetic diversity in the next generation, providing the feedstock for evolution. DNA double-strand breaks (DSBs), which are formed by the conserved Spo11 nuclease, initiate meiotic recombination. DSB mapping is thus an alternative to standard genetic analysis for determining where meiotic recombination occurs. DSBs have been most extensively mapped in budding yeast mutants that fail to remove Spo11 from break ends, blocking further recombination steps. Paradoxically, those studies indicated that DSBs are absent from large regions where recombination was known to occur. We developed a new DSB mapping method that purifies and analyzes the single-strand DNA formed at breaks after Spo11 removal. This new map shows that DSBs (and by inference, recombination) actually occur frequently throughout almost all of the budding yeast genome, in a distribution that is consistent with recombination's roles in chromosome pairing and in generating genetic diversity. This new mapping method will be useful for studying meiotic recombination and DNA damage repair in other organisms.
The authors developed a new method to detect DNA damage genome-wide, and they used it to show that meiotic recombination is more uniformly distributed in budding yeast than was previously believed.
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.
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.
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.
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.
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.
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.
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.
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.
Clustered DNA lesions are defined as ≥2 damage events within 20bp. Oxidised bases, abasic (AP) sites, single-strand breaks and double-strand breaks (DSBs) exist in radiation-induced clusters, and these lesions are more difficult to repair and can be more mutagenic than single lesions. Understanding clustered lesion repair is therefore important for the design of complementary treatments to enhance radiotherapy. Non-DSB-clustered lesions consisting of opposing AP sites can be converted to DSBs by base excision repair, and non-homologous end-joining (NHEJ) plays a role in repairing these DSBs. Artemis is an endonuclease that removes blocking groups from DSB termini during NHEJ. Hence, we hypothesised that Artemis plays a role in the processing of DSBs or complex DSBs generated from non-DSB-clustered lesions. We examined the repair of clusters containing two or three lesions in wild-type (WT) or Artemis-deficient (ART−/−) mouse fibroblasts using a reporter plasmid. Each cluster contained two opposing tetrahydrofurans (an AP site analogue), which AP endonuclease can convert to a DSB with blocked 5′ termini. Loss of Artemis did not decrease plasmid survival, but did result in more mutagenic repair with plasmids containing larger deletions. This increase in deletions did not occur with ClaI-linearised plasmid. Since Mre11 has been implicated in deletional NHEJ, we used small interfering RNA to reduce Mre11 in WT and ART−/− cells, but decreasing Mre11 did not change the size of deletions in the repair products. This work implicates Artemis in limiting the deletions introduced during repair of 5′-blocked termini DSBs generated from non-DSB-clustered lesions. Decreasing repair accuracy without decreasing repair capacity could result in mutated cells surviving irradiation. Inhibiting Artemis in normal cells could promote carcinogenesis, while in tumour cells enhanced mutagenic repair following irradiation could promote tumour recurrence.
The Mre11-Rad50-Xrs2 nuclease complex, together with Sae2, initiates the 5′-to-3′ resection of Double-Strand DNA Breaks (DSBs). Extended 3′ single stranded DNA filaments can be exposed from a DSB through the redundant activities of the Exo1 nuclease and the Dna2 nuclease with the Sgs1 helicase. In the absence of Sae2, Mre11 binding to a DSB is prolonged, the two DNA ends cannot be kept tethered, and the DSB is not efficiently repaired. Here we show that deletion of the yeast 53BP1-ortholog RAD9 reduces Mre11 binding to a DSB, leading to Rad52 recruitment and efficient DSB end-tethering, through an Sgs1-dependent mechanism. As a consequence, deletion of RAD9 restores DSB repair either in absence of Sae2 or in presence of a nuclease defective MRX complex. We propose that, in cells lacking Sae2, Rad9/53BP1 contributes to keep Mre11 bound to a persistent DSB, protecting it from extensive DNA end resection, which may lead to potentially deleterious DNA deletions and genome rearrangements.
DNA double strand breaks (DSBs) are among the most deleterious types of damage occurring in the genome, as failure to repair these lesions through either non-homologous-end-joining (NHEJ) or homologous recombination (HR) leads to genetic instability. The 5′ strand of a DSB can be nucleolytically degraded by several nucleases and associated factors, including Mre11, CtIP/Sae2, Exo1 and Dna2 together with Bloom helicase/Sgs1, through a finely regulated process called DSB resection. Once resection is initiated, error-prone NHEJ is prevented. Several findings suggest that DSB resection is a double-edged sword, if not finely regulated, since on one hand it is needed for faithful HR, but on the other it may lead to extensive DNA deletions associated with genome instability. Both in mammals and yeast, 53BP1/Rad9 protein binds near the lesion and counteracts the resection process, limiting the formation of ssDNA. By using S. cerevisiae as a model organism, here we show that Rad9 oligomers block the removal of hypo-active Mre11 protein from a persistent DSB, thus limiting initiation of resection and the recruitment of the recombination factor Rad52, in the absence of Sae2. Altogether, these findings pinpoint a critical role of 53BP1/Rad9 in balancing HR and NHEJ repair events throughout the cell cycle.
In budding yeast, an HO endonuclease-inducible double-strand break (DSB) is efficiently repaired by several homologous recombination (HR) pathways. In contrast to gene conversion (GC), where both ends of the DSB can recombine with the same template, break-induced replication (BIR) occurs when only the centromere-proximal end of the DSB can locate homologous sequences. Whereas GC results in a small patch of new DNA synthesis, BIR leads to a nonreciprocal translocation. The requirements for completing BIR are significantly different from those of GC, but both processes require 5′ to 3′ resection of DSB ends to create single-stranded DNA that leads to formation of a Rad51 filament required to initiate HR. Resection proceeds by two pathways dependent on Exo1 or the BLM homolog, Sgs1. We report that Exo1 and Sgs1 each inhibit BIR but have little effect on GC, while overexpression of either protein severely inhibits BIR. In contrast, overexpression of Rad51 markedly increases the efficiency of BIR, again with little effect on GC. In sgs1Δ exo1Δ strains, where there is little 5′ to 3′ resection, the level of BIR is not different from either single mutant; surprisingly, there is a two-fold increase in cell viability after HO induction whereby 40% of all cells survive by formation of a new telomere within a few kb of the site of DNA cleavage. De novo telomere addition is rare in wild-type, sgs1Δ, or exo1Δ cells. In sgs1Δ exo1Δ, repair by GC is severely inhibited, but cell viaiblity remains high because of new telomere formation. These data suggest that the extensive 5′ to 3′ resection that occurs before the initiation of new DNA synthesis in BIR may prevent efficient maintenance of a Rad51 filament near the DSB end. The severe constraint on 5′ to 3′ resection, which also abrogates activation of the Mec1-dependent DNA damage checkpoint, permits an unprecedented level of new telomere addition.
A chromosomal double-strand break (DSB) poses a severe threat to genome integrity, and budding yeast cells use several homologous recombination mechanisms to repair the break. In gene conversion (GC), both ends of the DSB share homology to an intact donor locus, and the break is repaired by copying the donor to create a small patch of new DNA synthesis. In break-induced replication (BIR), only one side of the DSB shares homology to a donor, and repair involves assembly of a recombination-dependent replication fork that copies sequences to the end of the template chromosome, yielding a nonreciprocal translocation. Both processes require that the DSB ends be resected by 5′ to 3′ exonucleases, involving several proteins or protein complexes, including Exo1 and Sgs1-Rmi1-Top3-Dna2. We report that ectopic BIR is inhibited independently by Sgs1 and Exo1 and that overexpression of Rad51 recombinase further improves BIR, while GC is largely unaffected. Surprisingly, when both Sgs1 and Exo1 are deleted, and resection is severely impaired, half of the cells acquire new telomeres rather than completing BIR or GC. New telomere addition appears to result from the lack of resection itself and from the fact that, without resection, the Mec1 (ATR) DNA damage checkpoint fails to inactivate the Pif1 helicase that discourages new telomere formation.
Immunoglobulin class switch recombination (CSR) occurs by an intrachromosomal deletion requiring generation of double-stranded DNA breaks (DSBs) in immunoglobulin switch region DNA. The initial steps of DSB formation have been elucidated: cytosine deamination by activation-induced cytidine deaminase (AID) and the generation of abasic sites by uracil-DNA glycosylase (UNG). We show that abasic sites are converted into single-strand breaks (SSBs) by apurinic/apyrimidinic endonucleases (APE1 and APE2). If SSBs are near to each other on opposite strands, they will generate DSBs; but if distal from each other, mismatch repair appears to be required to generate DSBs. The resulting S region DSBs occur at dC residues that are preferentially targeted by AID. We also investigate whether DNA polymerase β, which correctly repairs SSBs resulting from APE activity, attempts to repair the breaks during CSR. We find that although polymerase β does attempt to repair S region DNA breaks in switching B cells, the frequency of AID-instigated breaks appears to outnumber the SSBs repaired correctly by polymerase β, and thus some DSBs and mutations are generated. We also show that the S region DSBs are introduced and resolved during the G1 phase of the cell cycle.
antibody class switch; DNA recombination; activation induced cytidine deaminase
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.
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.
Topoisomerase inhibitors such as camptothecin and etoposide are used as anti-cancer drugs and induce double-strand breaks (DSBs) in genomic DNA in cycling cells. These DSBs are often covalently bound with polypeptides at the 3′ and 5′ ends. Such modifications must be eliminated before DSB repair can take place, but it remains elusive which nucleases are involved in this process. Previous studies show that CtIP plays a critical role in the generation of 3′ single-strand overhang at “clean” DSBs, thus initiating homologous recombination (HR)–dependent DSB repair. To analyze the function of CtIP in detail, we conditionally disrupted the CtIP gene in the chicken DT40 cell line. We found that CtIP is essential for cellular proliferation as well as for the formation of 3′ single-strand overhang, similar to what is observed in DT40 cells deficient in the Mre11/Rad50/Nbs1 complex. We also generated DT40 cell line harboring CtIP with an alanine substitution at residue Ser332, which is required for interaction with BRCA1. Although the resulting CtIPS332A/−/− cells exhibited accumulation of RPA and Rad51 upon DNA damage, and were proficient in HR, they showed a marked hypersensitivity to camptothecin and etoposide in comparison with CtIP+/−/− cells. Finally, CtIPS332A/−/−BRCA1−/− and CtIP+/−/−BRCA1−/− showed similar sensitivities to these reagents. Taken together, our data indicate that, in addition to its function in HR, CtIP plays a role in cellular tolerance to topoisomerase inhibitors. We propose that the BRCA1-CtIP complex plays a role in the nuclease-mediated elimination of oligonucleotides covalently bound to polypeptides from DSBs, thereby facilitating subsequent DSB repair.
Induction of double-strand breaks (DSBs) in chromosomal DNA effectively activates a program of cellular suicide and is widely used for chemotherapy on malignant cancer cells. Cells resist such therapies by quickly repairing the DSBs. Repair is carried out by two major DSB repair pathways, homologous recombination (HR) and nonhomologous end-joining. However, these pathways cannot join DSBs if their ends are chemically modified, as seen in the DSB ends that would arise after the prolonged treatment of the cells with topoisomerase inhibitors such as camptothecin and etoposide. These anti-cancer drugs can produce the polypeptides covalently attached to the 3′ or 5′ end of DSBs. It remains elusive which enzymes eliminate these chemical modifications prior to repair. We here show evidence that the BRCA1-CtIP complex plays a role in eliminating this chemical modification, thereby facilitating subsequent DSB repair. Thus, BRCA1 and CtIP have dual functions: their previously documented roles in HR and this newly identified function. This study contributes to our ability to predict the effectiveness of chemotherapeutic agents prior to their selection by evaluating the activity of individual repair factors. Accurate prediction is crucial, because chemotherapeutic agents that cause DNA damage have such strong side effects.
Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.
Double strand DNA breaks (DSBs) are dangerous chromosomal lesions that can lead to genome rearrangements, genetic instability, and cancer if not accurately repaired. Eukaryotes activate a surveillance mechanism, called DNA damage checkpoint, to arrest cell cycle progression and facilitate DNA repair. Several factors are physically recruited to DSBs, and specific kinases phosphorylate multiple targets leading to checkpoint activation. Budding yeast is a good model system to study checkpoint, and most of the factors involved in the DSBs response were originally characterized in this organism. Using the yeast Saccharomyces cerevisiae, we explored the functional role of polo kinase Cdc5 in regulating the DSB–induced checkpoint. Polo kinases have been previously involved in checkpoint inactivation in all the eukaryotes, and they are frequently overexpressed in cancer cells. We found that elevated levels of Cdc5 affect the cellular response to a DSB at different steps, altering DNA processing and overriding the signal triggered by checkpoint kinases. Our findings suggest that Cdc5 likely regulates multiple factors in response to a DSB and provide a rationale for a proteome-wide screening to identify targets of polo kinases in yeast and human cells. Such information may have a practical application to design specific molecular tools for cancer therapy. Two related papers published in PLoS Biology—by Vidanes et al., doi:10.1371/journal.pbio.1000286, and van Vugt et al., doi:10.1371/journal.pbio.1000287—similarly investigate the phenomenon of checkpoint adaptation/overriding.
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 sgs1Δ exo1Δ 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 sgs1Δ exo1Δ, 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 sgs1Δ exo1Δ 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.
Chromosomal breaks occur spontaneously or are induced by ionizing radiation and many chemotherapeutic drugs. DNA double-strand breaks are processed by nucleases and helicases in yeast and human to generate single-stranded DNA that is then used for repair by recombination with homologous chromosome. Single-stranded DNA at chromosomal breaks also constitutes a signal for cells to arrest cell cycle progression until the DNA damage is repaired. This study examines the consequences of elimination of enzymes that process chromosomal breaks to single-stranded DNA on the fidelity of repair and genome stability in the model organism yeast. Mutants deficient in these enzymes often fail to repair the breaks by homologous recombination and instead add new telomeres at the breaks. Formation of new telomeres is associated with partial loss of the chromosome arm distal from the break. Such chromosomal aberrations were frequently observed in tumor cells and are responsible for about 10% of human genomic disorders resulting from chromosomal abnormalities. We also observed that elimination of enzymes that process chromosomal breaks into single-stranded DNA greatly stimulates genome manipulation by gene targeting, suggesting that transformed DNA is also a substrate for degradation by these enzymes. We discuss the possibility of using a similar approach in mammalian cells where gene targeting is inaccurate and less efficient when compared to yeast.
Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) in haploid cells is generally restricted to S/G2 cell cycle phases, when DNA has been replicated and a sister chromatid is available as a repair template. This cell cycle specificity depends on cyclin-dependent protein kinases (Cdk1 in Saccharomyces cerevisiae), which initiate HR by promoting 5′–3′ nucleolytic degradation of the DSB ends. Whether Cdk1 regulates other HR steps is unknown. Here we show that yku70Δ cells, which accumulate single-stranded DNA (ssDNA) at the DSB ends independently of Cdk1 activity, are able to repair a DSB by single-strand annealing (SSA) in the G1 cell cycle phase, when Cdk1 activity is low. This ability to perform SSA depends on DSB resection, because both resection and SSA are enhanced by the lack of Rad9 in yku70Δ G1 cells. Furthermore, we found that interchromosomal noncrossover recombinants are generated in yku70Δ and yku70Δ rad9Δ G1 cells, indicating that DSB resection bypasses Cdk1 requirement also for carrying out these recombination events. By contrast, yku70Δ and yku70Δ rad9Δ cells are specifically defective in interchromosomal crossover recombination when Cdk1 activity is low. Thus, Cdk1 promotes DSB repair by single-strand annealing and noncrossover recombination by acting mostly at the resection level, whereas additional events require Cdk1-dependent regulation in order to generate crossover outcomes.
Homologous recombination (HR) provides an important mechanism to eliminate deleterious lesions, such as DNA double-strand breaks (DSBs). DSB repair by HR uses homologous DNA sequences as a template to form recombinants that are either crossover or noncrossover with regard to flanking parental sequences. Furthermore, a DSB flanked by direct DNA repeats can be repaired by another HR pathway called single-strand annealing (SSA). HR is generally confined to the S and G2 phases of the cell cycle, when DNA has been replicated and a sister chromatid is available as repair template. This cell cycle specificity depends on the activity of cyclin-dependent kinases (Cdks), which regulate initiation of HR by promoting nucleolytic degradation (resection) of the DSB ends. Whether Cdks regulate other HR steps is unknown. Here, we show that Saccharomyces cerevisiae Cdk1 has a dual function in HR: it promotes SSA and noncrossover recombination by regulating primarily the resection step, whereas it plays additional functions in allowing recombination accompanied by crossovers. As crossovers during mitotic cell growth have the potential for deleterious genome rearrangements when the sister chromatid is not used as repair template, this additional function of Cdk1 in promoting crossovers can provide another safety mechanism to ensure genome stability.
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.
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.
During the first meiotic prophase, programmed DNA double-strand breaks (DSBs) are distributed non randomly at hotspots along chromosomes, to initiate recombination. In all organisms, more DSBs are formed than crossovers (CO), the repair product that creates a physical link between homologs and allows their correct segregation. It is not known whether all DSB hotspots are also CO hotspots or if the CO/DSB ratio varies with the chromosomal location. Here, we investigated the variations in the CO/DSB ratio by mapping genome-wide the binding sites of the Zip3 protein during budding yeast meiosis. We show that Zip3 associates with DSB sites that are engaged in repair by CO, and Zip3 enrichment at DSBs reflects the DSB tendency to be repaired by CO. Moreover, the relative amount of Zip3 per DSB varies with the chromosomal location, and specific chromosomal features are associated with high or low Zip3 per DSB. This work shows that DSB hotspots are not necessarily CO hotspots and suggests that different categories of DSB sites may fulfill different functions.
For sexual reproduction, meiosis is an essential step ensuring the formation of haploid gametes from diploid precursors of the germline. This reduction in the genome's content is achieved through a specialized type of division, where a single round of DNA replication is followed by two successive rounds of chromosome segregation. The first round separates the homologs. For this to faithfully occur, homologous chromosomes pair with each other and experience recombination, catalyzed by the formation of programmed double-strand breaks (DSBs). Upon their repair, a subset of DSBs will generate crossovers, which result from an intermediate that creates a physical link between homologs and allows their correct segregation by the meiotic spindle. DSBs, as well as crossovers, do not occur randomly along chromosomes but at preferential places called hotspots. To ask if all DSB hotspots also give rise to high crossover frequency, we have systematically compared the map of DSBs with that of a protein, Zip3, which we show preferentially binds to DSB sites that are being repaired with a crossover. We discovered that several DSB hotspots rarely produce crossovers, meaning that the decision to repair a DSB with a crossover can be influenced by specific chromosomal features.
DNA double-strand breaks and chromosomal aberrations after treatment with N-alkylating agents likely arise as a result of replication fork collision with single-strand breaks generated during base excision repair.
Exposures that methylate DNA potently induce DNA double-strand breaks (DSBs) and chromosomal aberrations, which are thought to arise when damaged bases block DNA replication. Here, we demonstrate that DNA methylation damage causes DSB formation when replication interferes with base excision repair (BER), the predominant pathway for repairing methylated bases. We show that cells defective in the N-methylpurine DNA glycosylase, which fail to remove N-methylpurines from DNA and do not initiate BER, display strongly reduced levels of methylation-induced DSBs and chromosomal aberrations compared with wild-type cells. Also, cells unable to generate single-strand breaks (SSBs) at apurinic/apyrimidinic sites do not form DSBs immediately after methylation damage. In contrast, cells deficient in x-ray cross-complementing protein 1, DNA polymerase β, or poly (ADP-ribose) polymerase 1 activity, all of which fail to seal SSBs induced at apurinic/apyrimidinic sites, exhibit strongly elevated levels of methylation-induced DSBs and chromosomal aberrations. We propose that DSBs and chromosomal aberrations after treatment with N-alkylators arise when replication forks collide with SSBs generated during BER.
In mammalian meiotic prophase, the initial steps in repair of SPO11-induced DNA double-strand breaks (DSBs) are required to obtain stable homologous chromosome pairing and synapsis. The X and Y chromosomes pair and synapse only in the short pseudo-autosomal regions. The rest of the chromatin of the sex chromosomes remain unsynapsed, contains persistent meiotic DSBs, and the whole so-called XY body undergoes meiotic sex chromosome inactivation (MSCI). A more general mechanism, named meiotic silencing of unsynapsed chromatin (MSUC), is activated when autosomes fail to synapse. In the absence of SPO11, many chromosomal regions remain unsynapsed, but MSUC takes place only on part of the unsynapsed chromatin. We asked if spontaneous DSBs occur in meiocytes that lack a functional SPO11 protein, and if these might be involved in targeting the MSUC response to part of the unsynapsed chromatin. We generated mice carrying a point mutation that disrupts the predicted catalytic site of SPO11 (Spo11YF/YF), and blocks its DSB-inducing activity. Interestingly, we observed foci of proteins involved in the processing of DNA damage, such as RAD51, DMC1, and RPA, both in Spo11YF/YF and Spo11 knockout meiocytes. These foci preferentially localized to the areas that undergo MSUC and form the so-called pseudo XY body. In SPO11-deficient oocytes, the number of repair foci increased during oocyte development, indicating the induction of S phase-independent, de novo DNA damage. In wild type pachytene oocytes we observed meiotic silencing in two types of pseudo XY bodies, one type containing DMC1 and RAD51 foci on unsynapsed axes, and another type containing only RAD51 foci, mainly on synapsed axes. Taken together, our results indicate that in addition to asynapsis, persistent SPO11-induced DSBs are important for the initiation of MSCI and MSUC, and that SPO11-independent DNA repair foci contribute to the MSUC response in oocytes.
Meiosis is a special cell division that generates genetically divergent haploid germ cells. At the very beginning of this process, during meiotic prophase, the enzyme SPO11 generates hundreds of DNA double-strand breaks (DSBs). Meiotic DSBs are repaired via a mechanism that requires the presence of an intact homologous template. This repair process stimulates homologous chromosome pairing, and the formation of a protein complex that connects the paired chromosome axes, reaching a state called synapsis. Male mammals carry a pair of largely heterologous sex chromosomes, the X and Y, which show delayed DSB repair and extensive asynapsis. In addition, the X and Y chromosomes are transcriptionally silenced by a mechanism named Meiotic Sex Chromosome Inactivation (MSCI). This mechanism is a specialization of a more general silencing mechanism, named Meiotic Silencing of Unsynapsed Chromatin (MSUC), that is induced when any pairing problem between homologous chromosomes results in asynapsis, in male as well as female meiotic prophase cells. Here, we demonstrate that in addition to asynapsis, the persistent presence of DNA repair foci is a hallmark of meiotic silencing. In addition, we show that SPO11-independent DNA repair foci form during normal oocyte development. We propose that these foci represent sites of unrepaired DSBs that are capable of inducing transcriptional silencing, irrespective of synapsis.
DNA double-strand breaks (DSBs) are harmful lesions that arise mainly during replication. The choice of the sister chromatid as the preferential repair template is critical for genome integrity, but the mechanisms that guarantee this choice are unknown. Here we identify new genes with a specific role in assuring the sister chromatid as the preferred repair template. Physical analyses of sister chromatid recombination (SCR) in 28 selected mutants that increase Rad52 foci and inter-homolog recombination uncovered 8 new genes required for SCR. These include the SUMO/Ub-SUMO protease Wss1, the stress-response proteins Bud27 and Pdr10, the ADA histone acetyl-transferase complex proteins Ahc1 and Ada2, as well as the Hst3 and Hst4 histone deacetylase and the Rtt109 histone acetyl-transferase genes, whose target is histone H3 Lysine 56 (H3K56). Importantly, we use mutations in H3K56 residue to A, R, and Q to reveal that H3K56 acetylation/deacetylation is critical to promote SCR as the major repair mechanism for replication-born DSBs. The same phenotype is observed for a particular class of rad52 alleles, represented by rad52-C180A, with a DSB repair defect but a spontaneous hyper-recombination phenotype. We propose that specific Rad52 residues, as well as the histone H3 acetylation/deacetylation state of chromatin and other specific factors, play an important role in identifying the sister as the choice template for the repair of replication-born DSBs. Our work demonstrates the existence of specific functions to guarantee SCR as the main repair event for replication-born DSBs that can occur by two pathways, one Rad51-dependent and the other Pol32-dependent. A dysfunction can lead to genome instability as manifested by high levels of homolog recombination and DSB accumulation.
Double-strand breaks (DSBs) are among the most dangerous DNA lesions and can lead to genomic instability, a process associated with cancer and hereditary diseases. An important source of DSBs is replication, Sister Chromatid Recombination (SCR) being the main mechanism for DSB repair in dividing eukaryotic cells. SCR repair is error-free and uses the sister chromatid as template, generating an identical DNA sequence and therefore preventing genomic instability. In this work, we use an inverted-repeat assay with which we can physically detect SCR intermediates generated by the repair of a replication-born DSB. We hypothesized that SCR defects can result in an increase of recombination with the homologous chromosome, so we assayed SCR in 28 mutants previously described to increase homolog recombination. Our results describe 8 new genes involved in SCR, including functions such as histone acetylation/deacetylation, SUMO-Ubiquitin metabolism, and stress response, as well as an allele of RAD52. This demonstrates the importance of the choice of the sister chromatid as template for DSB repair and provides a broad vision of SCR as a tightly regulated process essential for genome integrity.
Homology-dependent repair of DNA double-strand breaks (DSBs) by gene conversion involves short tracts of DNA synthesis and limited loss of heterozygosity (LOH). For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome resulting in extensive LOH, a process called break-induced replication (BIR). We developed a BIR assay in Saccharomyces cerevisiae consisting of a plasmid with a telomere seeding sequence separated from sequence homologous to chromosome III by an I-SceI endonuclease recognition site. Following cleavage of the plasmid by I-SceI in vivo, de novo telomere synthesis occurs at one end of the vector, and the other end invades at the homologous sequence on chromosome III and initiates replication to the end of the chromosome to generate a stable chromosome fragment (CF). BIR was infrequent in wild-type cells due to degradation of the linearized vector. However, in the exo1Δ sgs1Δ mutant, which is defective in the 5′-3′ resection of DSBs, the frequency of BIR was increased by 39-fold. Extension of the invading end of the plasmid was detected by physical analysis two hours after induction of the I-SceI endonuclease in the wild-type exo1Δ, sgs1Δ, and exo1Δ sgs1Δ mutants, but fully repaired products were only visible in the exo1Δ sgs1Δ mutant. The inhibitory effect of resection was less in a plasmid-chromosome gene conversion assay, compared to BIR, and products were detected by physical assay in the wild-type strain. The rare chromosome rearrangements due to BIR template switching at repeated sequences were increased in the exo1Δ sgs1Δ mutant, suggesting that reduced resection can decrease the fidelity of homologous recombination.
DNA double-strand breaks (DSBs) can occur spontaneously in cells by defective DNA replication or are induced by various types of DNA damaging agents, such as those used in chemo- or radiation therapy. Failure to repair DSBs, or inappropriate repair, can result in chromosome loss or chromosome rearrangements, events associated with development of cancer cells. Typically, DNA DSBs have two ends that are reunited faithfully by copying from a homologous donor chromosome. For DSBs that present only one end, repair occurs by invasion into a homologous sequence followed by replication to the end of the chromosome, a process called break-induced replication (BIR). This repair pathway is thought to be suppressed at two-ended DSBs to prevent extensive loss of heterozygosity. Here, we describe a new assay to physically monitor BIR to see how this repair pathway differs from the repair of two-ended DSBs. We show this pathway is infrequent, but can be detected by eliminating factors (Exo1 and Sgs1) that degrade linear DNA. However, increased chromosome rearrangements were found in the resection-defective strain. We found the kinetics of strand invasion in two-ended and one-ended DSB repair were the same, suggesting that the distinction between these pathways occurs after initial repair steps have begun.
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 EndoG−/− B cells did not stem from altered B cell proliferation or apoptosis. Rather, it was associated with significantly reduced frequency of DSBs. Thus, our findings determine a role for EndoG in the generation of S region DSBs and CSR.
Activation-induced cytidine deaminase (AID); Antibody; B cell; Endonuclease G (EndoG); Class switch DNA recombination (CSR); Double-strand DNA break (DSB); Immunoglobulin (Ig); Switch region; Knockout mice
A DNA double strand break (DSB) is a highly toxic lesion, which can generate genetic instability and profound genome rearrangements. However, DSBs are required to generate diversity during physiological processes such as meiosis or the establishment of the immune repertoire. Thus, the precise regulation of a complex network of processes is necessary for the maintenance of genomic stability, allowing genetic diversity but protecting against genetic instability and its consequences on oncogenesis. Two main strategies are employed for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). HR is initiated by single-stranded DNA (ssDNA) resection and requires sequence homology with an intact partner, while NHEJ requires neither resection at initiation nor a homologous partner. Thus, resection is an pivotal step at DSB repair initiation, driving the choice of the DSB repair pathway employed. However, an alternative end-joining (A-EJ) pathway, which is highly mutagenic, has recently been described; A-EJ is initiated by ssDNA resection but does not require a homologous partner. The choice of the appropriate DSB repair system, for instance according the cell cycle stage, is essential for genome stability maintenance. In this context, controlling the initial events of DSB repair is thus an essential step that may be irreversible, and the wrong decision should lead to dramatic consequences. Here, we first present the main DSB repair mechanisms and then discuss the importance of the choice of the appropriate DSB repair pathway according to the cell cycle phase. In a third section, we present the early steps of DSB repair i.e., DSB signaling, chromatin remodeling, and the regulation of ssDNA resection. In the last part, we discuss the competition between the different DSB repair mechanisms. Finally, we conclude with the importance of the fine tuning of this network for genome stability maintenance and for tumor protection in fine.
DNA double strand break; Homologous recombination; Non homologous end joining; alternative end-joining; Resection; chromatin remodeling; genetic instability; genome rearrangements