Mechanisms restraining break‐induced replication at two‐ended DNA double‐strand breaks

DNA synthesis during homologous recombination is highly mutagenic and prone to template switches. Two‐ended DNA double‐strand breaks (DSBs) are usually repaired by gene conversion with a short patch of DNA synthesis, thus limiting the mutation load to the vicinity of the DSB. Single‐ended DSBs are repaired by break‐induced replication (BIR), which involves extensive and mutagenic DNA synthesis spanning up to hundreds of kilobases. It remains unknown how mutagenic BIR is suppressed at two‐ended DSBs. Here, we demonstrate that BIR is suppressed at two‐ended DSBs by proteins coordinating the usage of two ends of a DSB: (i) ssDNA annealing proteins Rad52 and Rad59 that promote second end capture, (ii) D‐loop unwinding helicase Mph1, and (iii) Mre11‐Rad50‐Xrs2 complex that promotes synchronous resection of two ends of a DSB. Finally, BIR is also suppressed when Sir2 silences a normally heterochromatic repair template. All of these proteins are particularly important for limiting BIR when recombination occurs between short repetitive sequences, emphasizing the significance of these mechanisms for species carrying many repetitive elements such as humans.     

Factors determining DNA double-strand break repair pathway choice in G2 phase

DNA non-homologous end joining (NHEJ) and homologous recombination (HR) function to repair DNA double-strand breaks (DSBs) in G2 phase with HR preferentially repairing heterochromatin-associated DSBs (HC-DSBs). Here, we examine the regulation of repair pathway usage at two-ended DSBs in G2. We identify the speed of DSB repair as a major component influencing repair pathway usage showing that DNA damage and chromatin complexity are factors influencing DSB repair rate and pathway choice. Loss of NHEJ proteins also slows DSB repair allowing increased resection. However, expression of an autophosphorylation-defective DNA-PKcs mutant, which binds DSBs but precludes the completion of NHEJ, dramatically reduces DSB end resection at all DSBs. In contrast, loss of HR does not impair repair by NHEJ although CtIP-dependent end resection precludes NHEJ usage. We propose that NHEJ initially attempts to repair DSBs and, if rapid rejoining does not ensue, then resection occurs promoting repair by HR. Finally, we identify novel roles for ATM in regulating DSB end resection; an indirect role in promoting KAP-1-dependent chromatin relaxation and a direct role in phosphorylating and activating CtIP.     

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

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

The chromatin remodeler Chd1 supports MRX and Exo1 functions in resection of DNA double-strand breaks

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) requires that the 5’-terminated DNA strands are resected to generate single-stranded DNA overhangs. This process is initiated by a short-range resection catalyzed by the MRX (Mre11-Rad50-Xrs2) complex, which is followed by a long-range step involving the nucleases Exo1 and Dna2. Here we show that the Saccharomyces cerevisiae ATP-dependent chromatin-remodeling protein Chd1 participates in both short- and long-range resection by promoting MRX and Exo1 association with the DSB ends. Furthermore, Chd1 reduces histone occupancy near the DSB ends and promotes DSB repair by HR. All these functions require Chd1 ATPase activity, supporting a role for Chd1 in the opening of chromatin at the DSB site to facilitate MRX and Exo1 processing activities.     

Loss of CHD1 causes DNA repair defects and enhances prostate cancer therapeutic responsiveness

The CHD1 gene, encoding the chromo‐domain helicase DNA‐binding protein‐1, is one of the most frequently deleted genes in prostate cancer. Here, we examined the role of CHD1 in DNA double‐strand break (DSB) repair in prostate cancer cells. We show that CHD1 is required for the recruitment of CtIP to chromatin and subsequent end resection during DNA DSB repair. Our data support a role for CHD1 in opening the chromatin around the DSB to facilitate the recruitment of homologous recombination (HR) proteins. Consequently, depletion of CHD1 specifically affects HR‐mediated DNA repair but not non‐homologous end joining. Together, we provide evidence for a previously unknown role of CHD1 in DNA DSB repair via HR and show that CHD1 depletion sensitizes cells to PARP inhibitors, which has potential therapeutic relevance. Our findings suggest that CHD1 deletion, like BRCA1/2 mutation in ovarian cancer, may serve as a marker for prostate cancer patient stratification and the utilization of targeted therapies such as PARP inhibitors, which specifically target tumors with HR defects.     

Mre11 and Exo1 contribute to the initiation and processivity of resection at meiotic double-strand breaks made independently of Spo11

During meiosis DNA double-strand breaks (DSBs) are induced and repaired by homologous recombination to create gene conversion and crossover products. Mostly these DSBs are made by Spo11, which covalently binds to the DSB ends. More rarely in Saccharomyces cerevisiae, other meiotic DSBs are formed by self-homing endonucleases such as VDE, which is site specific and does not covalently bind to the DSB ends. We have used experimentally located VDE-DSB sites to analyse an intermediate step in homologous recombination, resection of the single-strand ending 5' at the DSB site. Analysis of strains with different mutant alleles of MRE11 (mre11-58S and mre11-H125N) and deleted for EXO1 indicated that these two nucleases make significant contributions to repair of VDE-DSBs. Physical analysis of single-stranded repair intermediates indicates that efficient initiation and processivity of resection at VDE-DSBs require both Mre11 and Exo1, with loss of function for either protein causing severe delay in resection. We propose that these experiments model what happens at Spo11-DSBs after removal of the covalently bound protein, and that Mre11 and Exo1 are the major nucleases involved in creating resection tracts of widely varying lengths typical of meiotic recombination.     

The complexity of DNA double strand breaks is a critical factor enhancing end-resection

DNA double strand breaks (DSBs) induced by ionizing radiation (IR) are deleterious damages. Two major pathways repair DSBs in human cells, DNA non-homologous end-joining (NHEJ) and homologous recombination (HR). It has been suggested that the balance between the two repair pathways varies depending on the chromatin structure surrounding the damage site and/or the complexity of damage at the DNA break ends. Heavy ion radiation is known to induce complex-type DSBs, and the efficiency of NHEJ in repairing these DSBs was shown to be diminished. Taking advantage of the ability of high linear energy transfer (LET) radiation to produce complex DSBs effectively, we investigated how the complexity of DSB end structure influences DNA damage responses. An early step in HR is the generation of 3′-single strand DNA (SSD) via a process of DNA end resection that requires CtIP. To assess this process, we analyzed the level of phosphorylated CtIP, as well as RPA phosphorylation and focus formation, which occur on the exposed SSD. We show that complex DSBs efficiently activate DNA end resection. After heavy ion beam irradiation, resection signals appear both in the vicinity of heterochromatic areas, which is also observed after X-irradiation, and additionally in euchromatic areas. Consequently, ∼85% of complex DSBs are subjected to resection in heavy ion particle tracks. Furthermore, around 20–40% of G1 cells exhibit resection signals. Taken together, our observations reveal that the complexity of DSB ends is a critical factor regulating the choice of DSB repair pathway and drastically alters the balance toward resection-mediated rejoining. As demonstrated here, studies on DNA damage responses induced by heavy ion radiation provide an important tool to shed light on mechanisms regulating DNA end resection.     

Both CRISPR/Cas‐based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana

Engineered nucleases can be used to induce site‐specific double‐strand breaks (DSBs) in plant genomes. Thus, homologous recombination (HR) can be enhanced and targeted mutagenesis can be achieved by error‐prone non‐homologous end‐joining (NHEJ). Recently, the bacterial CRISPR/Cas9 system was used for DSB induction in plants to promote HR and NHEJ. Cas9 can also be engineered to work as a nickase inducing single‐strand breaks (SSBs). Here we show that only the nuclease but not the nickase is an efficient tool for NHEJ‐mediated mutagenesis in plants. We demonstrate the stable inheritance of nuclease‐induced targeted mutagenesis events in the ADH1 and TT4 genes of Arabidopsis thaliana at frequencies from 2.5 up to 70.0%. Deep sequencing analysis revealed NHEJ‐mediated DSB repair in about a third of all reads in T1 plants. In contrast, applying the nickase resulted in the reduction of mutation frequency by at least 740‐fold. Nevertheless, the nickase is able to induce HR at similar efficiencies as the nuclease or the homing endonuclease I–SceI. Two different types of somatic HR mechanisms, recombination between tandemly arranged direct repeats as well as gene conversion using the information on an inverted repeat could be enhanced by the nickase to a similar extent as by DSB‐inducing enzymes. Thus, the Cas9 nickase has the potential to become an important tool for genome engineering in plants. It should not only be applicable for HR‐mediated gene targeting systems but also by the combined action of two nickases as DSB‐inducing agents excluding off‐target effects in homologous genomic regions.     

Mre11 and Ku regulation of double-strand break repair by gene conversion and break-induced replication

The yeast Mre11-Rad50-Xrs2 (MRX) and Ku complexes regulate single-strand resection at DNA double-strand breaks (DSB), a key early step in homologous recombination (HR). A prior plasmid gap repair study showed that mre11 mutations, which slow single-strand resection, reduce gene conversion tract lengths and the frequency of associated crossovers. Here we tested whether mre11Delta or nuclease-defective mre11 mutations reduced gene conversion tract lengths during HR between homologous chromosomes in diploid yeast. We found that mre11 mutations reduced the efficiency of HR but did not reduce tract lengths or crossovers, despite substantially reduced end-resection at the test (ura3) locus. End-resection is increased in yku70Delta, but this change also had no effect on tract lengths. Thus, heteroduplex formation and tract lengths are not regulated by the extent of end-resection during DSB repair in a chromosomal context. In a plasmid-chromosome DSB repair assay, tract lengths were again similar in wild-type and mre11Delta, but they were reduced in mre11Delta in a gap repair assay. These results indicate that tract lengths are not affected by the extent of end processing when broken ends can invade nearby sites, perhaps because MRX coordination of the two broken ends is dispensable when ends invade nearby sites. Although HR outcome was largely unaffected in mre11 mutants, break-induced replication (BIR) and chromosome loss increased, suggesting that Mre11 function in mitotic HR is limited to early HR stages. Interestingly, yku70Delta suppressed BIR in mre11 mutants. BIR is also elevated in rad51 mutants, but yku70Delta did not suppress BIR in a rad51 background. These results indicate that Mre11 functions in Rad51-independent BIR, and that Ku functions in Rad51-dependent BIR.     

Distance from the chromosome end determines the efficiency of double strand break repair in subtelomeres of haploid yeast

Double strand break (DSB) repair plays an important role in chromosome evolution. We have investigated the fate of DSBs as a function of their location along the yeast chromosome XI, in a system where no conventional homologous recombination can occur. We report that the relative frequency of non-homologous endjoining (NHEJ), which is the exclusive mode of DSB repair in the internal chromosomal portion, decreases gradually towards the telomere, keeping the absolute frequency nearly constant, and that other repair mechanisms, which generally involve the loss of the distal chromosomal fragment, appear in subtelomeric regions. Distance of the DSB from chromosome ends plays a critical role in the global frequency of these repair mechanisms. Direct telomere additions are rare, and other events such as break-induced replication, plasmid incorporation, and gene conversion, involve acquisition of heterologous sequences. Therefore, in subtelomeric regions, cell survival to DSBs is higher and alternative modes of repair allow new genomic combinations to be generated. Furthermore, subtelomeric rearrangements depend on the recombination process, which, unexpectedly, also promotes the joining of heterologous sequences. Finally, we report that the Rad52 protein increases the efficiency of NHEJ.     

Mechanisms and regulation of DNA end resection

DNA double‐strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair these lesions can lead to genomic instability. DSBs can arise accidentally at unpredictable locations into the genome, but they are also normal intermediates in meiotic recombination. Moreover, the natural ends of linear chromosomes resemble DSBs. Although intrachromosomal DNA breaks are potent stimulators of the DNA damage response, the natural ends of linear chromosomes are packaged into protective structures called telomeres that suppress DNA repair/recombination activities. Although DSBs and telomeres are functionally different, they both undergo 5′–3′ nucleolytic degradation of DNA ends, a process known as resection. The resulting 3′‐single‐stranded DNA overhangs enable repair of DSBs by homologous recombination (HR), whereas they allow the action of telomerase at telomeres. The molecular activities required for DSB and telomere end resection are similar, indicating that the initial steps of HR and telomerase‐mediated elongation are related. Resection of both DSBs and telomeres must be tightly regulated in time and space to ensure genome stability and cell survival.     

Rice RAD51 paralogs play essential roles in somatic homologous recombination for DNA repair

Synthesis‐dependent strand annealing (SDSA) and single‐strand annealing (SSA) are the two main homologous recombination (HR) pathways in double‐strand break (DSB) repair. The involvement of rice RAD51 paralogs in HR is well known in meiosis, although the molecular mechanism in somatic HR remains obscure. Loss‐of‐function mutants of rad51 paralogs show increased sensitivity to the DSB‐inducer bleomycin, which results in greatly compromised somatic recombination efficiencies (xrcc3 in SDSA, rad51b and xrcc2 in SSA, rad51c and rad51d in both). Using immunostaining, we found that mutations in RAD51 paralogs (XRCC3, RAD51C, or RAD51D) lead to tremendous impairment in RAD51 focus formation at DSBs. Intriguingly, the RAD51C mutation has a strong effect on the protein loading of its partners (XRCC3 and RAD51B) at DSBs, which is similar to the phenomenon observed in the case of blocking PI3K‐like kinases in wild‐type plant. We conclude that the rice CDX3 complex acts in SDSA recombination while the BCDX2 complex acts in SSA recombination in somatic DSB repair. Importantly, RAD51C serves as a fulcrum for the local recruitment of its partners (XRCC3 for SDSA and RAD51B for SSA) and is positively modulated by PI3K‐like kinases to facilitate both the SDSA and SSA pathways in RAD51 paralog‐dependent somatic HR.     

The MRN complex promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes

Homologous recombination dominates as the major form of DNA repair in Trypanosoma brucei, and is especially important for recombination of the subtelomeric variant surface glycoprotein during antigenic variation. RAD50, a component of the MRN complex (MRE11, RAD50, NBS1), is central to homologous recombination through facilitating resection and governing the DNA damage response. The function of RAD50 in trypanosomes is untested. Here we report that RAD50 and MRE11 are required for RAD51-dependent homologous recombination and phosphorylation of histone H2A following a DNA double strand break (DSB), but neither MRE11 nor RAD50 substantially influence DSB resection at a chromosome-internal locus. In addition, we reveal intrinsic separation-of-function between T. brucei RAD50 and MRE11, with only RAD50 suppressing DSB repair using donors with short stretches of homology at a subtelomeric locus, and only MRE11 directing DSB resection at the same locus. Finally, we show that loss of either MRE11 or RAD50 causes a greater diversity of expressed VSG variants following DSB repair. We conclude that MRN promotes stringent homologous recombination at subtelomeric loci and restrains antigenic variation.     

The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny

The CRISPR/Cas nuclease is becoming a major tool for targeted mutagenesis in eukaryotes by inducing double‐strand breaks (DSBs) at pre‐selected genomic sites that are repaired by non‐homologous end joining (NHEJ) in an error‐prone way. In plants, it could be demonstrated that the Cas9 nuclease is able to induce heritable mutations in Arabidopsis thaliana and rice. Gene targeting (GT) by homologous recombination (HR) can also be induced by DSBs. Using a natural nuclease and marker genes, we previously developed an in planta GT strategy in which both a targeting vector and targeting locus are activated simultaneously via DSB induction during plant development. Here, we demonstrate that this strategy can be used for natural genes by CRISPR/Cas‐mediated DSB induction. We were able to integrate a resistance cassette into the ADH1 locus of A. thaliana via HR. Heritable events were identified using a PCR‐based genotyping approach, characterised by Southern blotting and confirmed on the sequence level. A major concern is the specificity of the CRISPR/Cas nucleases. Off‐target effects might be avoided using two adjacent sgRNA target sequences to guide the Cas9 nickase to each of the two DNA strands, resulting in the formation of a DSB. By amplicon deep sequencing, we demonstrate that this Cas9 paired nickase strategy has a mutagenic potential comparable with that of the nuclease, while the resulting mutations are mostly deletions. We also demonstrate the stable inheritance of such mutations in A. thaliana.     

Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.     

Subtelomeric regions in mammalian cells are deficient in DNA double-strand break repair

We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.     

Persistently bound Ku at DNA ends attenuates DNA end resection and homologous recombination

DNA double strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). The DNA cell cycle stage and resection of the DSB ends are two key mechanisms which are believed to push DSB repair to the HR pathway. Here, we show that the NHEJ factor Ku80 associates with DSBs in S phase, when HR is thought to be the preferred repair pathway, and its dynamics/kinetics at DSBs is similar to those observed for Ku80 in non-S phase in mammalian cells. A Ku homolog from Mycobacterium tuberculosis binds to and is retained at DSBs in S phase and was used as a tool to determine if blocking DNA ends affects end resection and HR in mammalian cells. A decrease in DNA end resection, as marked by IR-induced RPA, BrdU, and Rad51 focus formation, and HR are observed when Ku deficient rodent cells are complemented with Mt-Ku. Together, this data suggests that Ku70/80 binds to DSBs in all cell cycle stages and is likely actively displaced from DSB ends to free the DNA ends for DNA end resection and thus HR to occur.     

DNA End Resection:Facts and Mechanisms

DNA double-strand breaks (DSBs), which arise following exposure to a number of endogenous and exogenous agents, can be repaired by either the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways in eukaryotic cells. A vital step in HR repair is DNA end resection, which generates a long 30 single-stranded DNA (ssDNA) tail that can invade the homologous DNA strand. The generation of 30 ssDNA is not only essential for HR repair, but also promotes activation of the ataxia telangiectasia and Rad3-related protein (ATR). Multiple fac-tors, including the MRN/X complex, C-terminal-binding protein interacting protein (CtIP)/Sae2, exonuclease 1 (EXO1), Bloom syndrome protein (BLM)/Sgs1, DNA2 nuclease/helicase, and several chromatin remodelers, cooperate to complete the process of end resection. Here we review the basic machinery involved in DNA end resection in eukaryotic cells.