To trim or not to trim: Progression and control of DSB end resection

DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage, since they can lead to genome instability and chromosome rearrangements, which are hallmarks of cancer cells. To face this kind of lesion, eukaryotic cells developed two alternative repair pathways, homologous recombination (HR) and non-homologous end joining (NHEJ). Repair pathway choice is influenced by the cell cycle phase and depends upon the 5′-3′ nucleolytic processing of the break ends, since the generation of ssDNA tails strongly stimulates HR and inhibits NHEJ. A large amount of work has elucidated the key components of the DSBs repair machinery and how this crucial process is finely regulated. The emerging view suggests that besides endo/exonucleases and helicases activities required for end resection, molecular barrier factors are specifically loaded in the proximity of the break, where they physically or functionally limit DNA degradation, preventing excessive accumulation of ssDNA, which could be threatening for cell survival.     

End Resection at Double-Strand Breaks: Mechanism and Regulation

RecA/Rad51 catalyzed pairing of homologous DNA strands, initiated by polymerization of the recombinase on single-stranded DNA (ssDNA), is a universal feature of homologous recombination (HR). Generation of ssDNA from a double-strand break (DSB) requires nucleolytic degradation of the 5′-terminated strands to generate 3′-ssDNA tails, a process referred to as 5′–3′ end resection. The RecBCD helicase–nuclease complex is the main end-processing machine in Gram-negative bacteria. Mre11-Rad50 and Mre11-Rad50-Xrs2/Nbs1 can play a direct role in end resection in archaea and eukaryota, respectively, by removing end-blocking lesions and act indirectly by recruiting the helicases and nucleases responsible for extensive resection. In eukaryotic cells, the initiation of end resection has emerged as a critical regulatory step to differentiate between homology-dependent and end-joining repair of DSBs.DSBs can arise accidentally during normal cell metabolism or after exposure of cells to DNA-damaging agents, and also serve as intermediates in a number of programmed recombination events in eukaryotic cells (Mehta and Haber 2014). The repair of DSBs is critical for maintenance of genome integrity, and misrepair, or failure to repair, is associated with chromosome rearrangements, chromosome loss, or even cell death. Both prokaryotic and eukaryotic cells have evolved elaborate mechanisms for the recognition and repair of DSBs. The two predominant repair mechanisms are HR and non-homologous end joining (NHEJ). HR relies on the presence of an intact homologous duplex to template repair of the broken strands, whereas NHEJ repairs DSBs by direct ligation of the DNA ends. For DSBs to be repaired by HR, the ends must first be degraded to generate long 3′-ssDNA tails, a process referred to as 5′–3′ end resection. The 3′-ssDNA tails are then bound by a member of the RecA/Rad51 family of proteins to initiate homologous pairing and serve as primers for DNA synthesis following strand invasion. Strand invasion intermediates are further processed by helicases and/or nucleases (Bizard and Hickson 2014; Wyatt and West 2014), and ultimately by gap-filling DNA synthesis and ligation, to generate mature recombinant products. The DNA end-resection step of HR is conserved in all domains of life, but the mechanisms used for generating ssDNA are distinct. Here, we review the basic machinery for DNA end resection in bacteria, archaea, and eukaryota and the regulation of end resection in eukaryotic cells.     

Mechanism and regulation of DNA end resection in eukaryotes

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic degradation of the 5′-terminated strands in a process termed end resection. End resection generates 3′-single-stranded DNA tails, substrates for Rad51 to catalyze homologous pairing and DNA strand exchange, and for activation of the DNA damage checkpoint. The commonly accepted view is that end resection occurs by a two-step mechanism. In the first step, Sae2/CtIP activates the Mre11–Rad50–Xrs2/Nbs1 (MRX/N) complex to endonucleolytically cleave the 5′-terminated DNA strands close to break ends, and in the second step Exo1 and/or Dna2 nucleases extend the resected tracts to produce long 3′-ssDNA-tailed intermediates. Initiation of resection commits a cell to repair a DSB by HR because long ssDNA overhangs are poor substrates for non-homologous end joining (NHEJ). Thus, the initiation of end resection has emerged as a critical control point for repair pathway choice. Here, I review recent studies on the mechanism of end resection and how this process is regulated to ensure the most appropriate repair outcome.     

FBH1 Helicase Disrupts RAD51 Filaments in Vitro and Modulates Homologous Recombination in Mammalian Cells

Efficient repair of DNA double strand breaks and interstrand cross-links requires the homologous recombination (HR) pathway, a potentially error-free process that utilizes a homologous sequence as a repair template. A key player in HR is RAD51, the eukaryotic ortholog of bacterial RecA protein. RAD51 can polymerize on DNA to form a nucleoprotein filament that facilitates both the search for the homologous DNA sequences and the subsequent DNA strand invasion required to initiate HR. Because of its pivotal role in HR, RAD51 is subject to numerous positive and negative regulatory influences. Using a combination of molecular genetic, biochemical, and single-molecule biophysical techniques, we provide mechanistic insight into the mode of action of the FBH1 helicase as a regulator of RAD51-dependent HR in mammalian cells. We show that FBH1 binds directly to RAD51 and is able to disrupt RAD51 filaments on DNA through its ssDNA translocase function. Consistent with this, a mutant mouse embryonic stem cell line with a deletion in the FBH1 helicase domain fails to limit RAD51 chromatin association and shows hyper-recombination. Our data are consistent with FBH1 restraining RAD51 DNA binding under unperturbed growth conditions to prevent unwanted or unscheduled DNA recombination.     

Focus: 50 Years of DNA Repair: The Yale Symposium Reports: Investigations of Homologous Recombination Pathways and Their Regulation

The DNA double-strand break (DSB), arising from exposure to ionizing radiation or various chemotherapeutic agents or from replication fork collapse, is among the most dangerous of chromosomal lesions. DSBs are highly cytotoxic and can lead to translocations, deletions, duplications, or mutations if mishandled. DSBs are eliminated by either homologous recombination (HR), which uses a homologous template to guide accurate repair, or by nonhomologous end joining (NHEJ), which simply rejoins the two broken ends after damaged nucleotides have been removed. HR generates error-free repair products and is also required for generating chromosome arm crossovers between homologous chromosomes in meiotic cells. The HR reaction includes several distinct steps: resection of DNA ends, homologous DNA pairing, DNA synthesis, and processing of HR intermediates. Each occurs in a highly regulated fashion utilizing multiple protein factors. These steps are being elucidated using a combination of genetic tools, cell-based assays, and in vitro reconstitution with highly purified HR proteins. In this review, we summarize contributions from our laboratory at Yale University in understanding HR mechanisms in eukaryotic cells.     

Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks

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.     

DNA-dependent Protein Kinase Regulates DNA End Resection in Concert with Mre11-Rad50-Nbs1 (MRN) and Ataxia Telangiectasia-mutated (ATM)

The resection of DNA double strand breaks initiates homologous recombination (HR) and is critical for genomic stability. Using direct measurement of resection in human cells and reconstituted assays of resection with purified proteins in vitro, we show that DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a classic nonhomologous end joining factor, antagonizes double strand break resection by blocking the recruitment of resection enzymes such as exonuclease 1 (Exo1). Autophosphorylation of DNA-PKcs promotes DNA-PKcs dissociation and consequently Exo1 binding. Ataxia telangiectasia-mutated kinase activity can compensate for DNA-PKcs autophosphorylation and promote resection under conditions where DNA-PKcs catalytic activity is inhibited. The Mre11-Rad50-Nbs1 (MRN) complex further stimulates resection in the presence of Ku and DNA-PKcs by recruiting Exo1 and enhancing DNA-PKcs autophosphorylation, and it also inhibits DNA ligase IV/XRCC4-mediated end rejoining. This work suggests that, in addition to its key role in nonhomologous end joining, DNA-PKcs also acts in concert with MRN and ataxia telangiectasia-mutated to regulate resection and thus DNA repair pathway choice.     

Break dosage, cell cycle stage and DNA replication influence DNA double strand break response

DNA double strand breaks (DSBs) can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HR). HR requires nucleolytic degradation of 5' DNA ends to generate tracts of single-stranded DNA (ssDNA), which are also important for the activation of DNA damage checkpoints. Here we describe a quantitative analysis of DSB processing in the budding yeast Saccharomyces cerevisiae. We show that resection of an HO endonuclease-induced DSB is less extensive than previously estimated and provide evidence for significant instability of the 3' ssDNA tails. We show that both DSB resection and checkpoint activation are dose-dependent, especially during the G1 phase of the cell cycle. During G1, processing near the break is inhibited by competition with NHEJ, but extensive resection is regulated by an NHEJ-independent mechanism. DSB processing and checkpoint activation are more efficient in G2/M than in G1 phase, but are most efficient at breaks encountered by DNA replication forks during S phase. Our findings identify unexpected complexity of DSB processing and its regulation, and provide a framework for further mechanistic insights.     

Human Ku70/80 protein blocks exonuclease 1-mediated DNA resection in the presence of human Mre11 or Mre11/Rad50 protein complex

DNA double strand breaks (DSB) are repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). Recent genetic data in yeast shows that the choice between these two pathways for the repair of DSBs is via competition between the NHEJ protein, Ku, and the HR protein, Mre11/Rad50/Xrs2 (MRX) complex. To study the interrelationship between human Ku and Mre11 or Mre11/Rad50 (MR), we established an in vitro DNA end resection system using a forked model dsDNA substrate and purified human Ku70/80, Mre11, Mre11/Rad50, and exonuclease 1 (Exo1). Our study shows that the addition of Ku70/80 blocks Exo1-mediated DNA end resection of the forked dsDNA substrate. Although human Mre11 and MR bind to the forked double strand DNA, they could not compete with Ku for DNA ends or actively mediate the displacement of Ku from the DNA end either physically or via its exonuclease or endonuclease activity. Our in vitro studies show that Ku can block DNA resection and suggest that Ku must be actively displaced for DNA end processing to occur and is more complicated than the competition model established in yeast.     

MRE11-RAD50-NBS1 Complex Dictates DNA Repair Independent of H2AX

DNA double-strand breaks (DSBs) represent one of the most serious forms of DNA damage that can occur in the genome. Here, we show that the DSB-induced signaling cascade and homologous recombination (HR)-mediated DSB repair pathway can be genetically separated. We demonstrate that the MRE11-RAD50-NBS1 (MRN) complex acts to promote DNA end resection and the generation of single-stranded DNA, which is critically important for HR repair. These functions of the MRN complex can occur independently of the H2AX-mediated DNA damage signaling cascade, which promotes stable accumulation of other signaling and repair proteins such as 53BP1 and BRCA1 to sites of DNA damage. Nevertheless, mild defects in HR repair are observed in H2AX-deficient cells, suggesting that the H2AX-dependent DNA damage-signaling cascade assists DNA repair. We propose that the MRN complex is responsible for the initial recognition of DSBs and works together with both CtIP and the H2AX-dependent DNA damage-signaling cascade to facilitate repair by HR and regulate DNA damage checkpoints.     

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

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

Control of DNA end resection by yeast Hmo1p affects efficiency of DNA end-joining

The primary pathways for DNA double strand break (DSB) repair are homologous recombination (HR) and non-homologous end–joining (NHEJ). The choice between HR and NHEJ is influenced by the extent of DNA end resection, as extensive resection is required for HR but repressive to NHEJ. Conversely, association of the DNA end-binding protein Ku, which is integral to classical NHEJ, inhibits resection. In absence of key NHEJ components, a third repair pathway is exposed; this alternative-end joining (A-EJ) is a highly error-prone process that uses micro-homologies at the breakpoints and is initiated by DNA end resection. In Saccharomyces cerevisiae, the high mobility group protein Hmo1p has been implicated in controlling DNA end resection, suggesting its potential role in repair pathway choice. Using a plasmid end-joining assay, we show here that absence of Hmo1p results in reduced repair efficiency and accuracy, indicating that Hmo1p promotes end-joining; this effect is only observed on DNA with protruding ends. Notably, inhibition of DNA end resection in an hmo1Δ strain restores repair efficiency to the levels observed in wild-type cells. In absence of Ku, HMO1 deletion also reduces repair efficiency further, while inhibition of resection restores repair efficiency to the levels observed in kuΔ. We suggest that Hmo1p functions to control DNA end resection, thereby preventing error-prone A-EJ repair and directing repairs towards classical NHEJ. The very low efficiency of DSB repair in kuΔhmo1Δ cells further suggests that excessive DNA resection is inhibitory for A-EJ.     

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.     

SHLD2/FAM35A co‐operates with REV7 to coordinate DNA double‐strand break repair pathway choice

DNA double‐strand breaks (DSBs) can be repaired by two major pathways: non‐homologous end‐joining (NHEJ) and homologous recombination (HR). DNA repair pathway choice is governed by the opposing activities of 53BP1, in complex with its effectors RIF1 and REV7, and BRCA1. However, it remains unknown how the 53BP1/RIF1/REV7 complex stimulates NHEJ and restricts HR to the S/G2 phases of the cell cycle. Using a mass spectrometry (MS)‐based approach, we identify 11 high‐confidence REV7 interactors and elucidate the role of SHLD2 (previously annotated as FAM35A and RINN2) as an effector of REV7 in the NHEJ pathway. FAM35A depletion impairs NHEJ‐mediated DNA repair and compromises antibody diversification by class switch recombination (CSR) in B cells. FAM35A accumulates at DSBs in a 53BP1‐, RIF1‐, and REV7‐dependent manner and antagonizes HR by limiting DNA end resection. In fact, FAM35A is part of a larger complex composed of REV7 and SHLD1 (previously annotated as C20orf196 and RINN3), which promotes NHEJ and limits HR. Together, these results establish SHLD2 as a novel effector of REV7 in controlling the decision‐making process during DSB repair.     

Repair-specific functions of replication protein A

Replication protein A (RPA), the major eukaryotic single-strand DNA (ssDNA)-binding protein, is essential for replication, repair, recombination, and checkpoint activation. Defects in RPA-associated cellular activities lead to genomic instability, a major factor in the pathogenesis of cancer and other diseases. ssDNA binding activity is primarily mediated by two domains in the 70-kDa subunit of the RPA complex. These ssDNA interactions are mediated by a combination of polar residues and four conserved aromatic residues. Mutation of the aromatic residues causes a modest decrease in binding to long (30-nucleotide) ssDNA fragments but results in checkpoint activation and cell cycle arrest in cells. We have used a combination of biochemical analysis and knockdown replacement studies in cells to determine the contribution of these aromatic residues to RPA function. Cells containing the aromatic residue mutants were able to progress normally through S-phase but were defective in DNA repair. Biochemical characterization revealed that mutation of the aromatic residues severely decreased binding to short ssDNA fragments less than 20 nucleotides long. These data indicate that altered binding of RPA to short ssDNA intermediates causes a defect in DNA repair but not in DNA replication. These studies show that cells require different RPA functions in DNA replication and DNA repair.     

Distinct Cdk1 requirements during single-strand annealing, noncrossover, and crossover recombination

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.     

Cell Cycle-Dependent Regulation of Double-Strand Break Repair: A Role for the CDK

DNA Double-Strand Breaks (DSBs) are dangerous lesions that can lead to genomic instability and to cell death. Eukaryotic cells repair DSBs either by non-homologous end joining (NHEJ) or by homologous recombination (HR). Recent work has allowed to study the ability of yeast cells to repair a single, chromosomal DSB, at different stages of the cell cycle. Yeast cells repair the broken chromosome during the G1 stage only by NHEJ, whereas HR is the mechanism of choice during the rest of the cell cycle. HR does not require duplicated chromatids or passage through S-phase. Control over the fate of the broken chromosome is exerted by Clb-CDK activity, which is required to carry out the first step of HR, ssDNA resection. Similar results in other organisms suggest that this control is a conserved feature in all eukaryotes.     

Synthetic viability genomic screening defines Sae2 function in DNA repair

DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.     

Excess single-stranded DNA inhibits meiotic double-strand break repair

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.