Smc5/6 maintains stalled replication forks in a recombination‐competent conformation

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis‐segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination‐dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome‐wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage‐induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination‐competent conformation primed for replication restart.     

Spatial separation between replisome‐ and template‐induced replication stress signaling

Polymerase‐blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single‐stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re‐priming downstream of lesions can give rise to daughter‐strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9‐dependent mechanism of damage signaling is distinct from the Mrc1‐dependent, fork‐associated response to replication stress induced by conditions such as nucleotide depletion or replisome‐inherent problems, but reminiscent of replication‐independent checkpoint activation by single‐stranded DNA. Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase‐blocking lesions mainly emanates from Exo1‐processed, postreplicative daughter‐strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome‐ versus template‐induced checkpoint signaling.     

Differential regulation of homologous recombination at DNA breaks and replication forks by the Mrc1 branch of the S‐phase checkpoint

The Rad52 pathway has a central function in the recombinational repair of chromosome breaks and in the recovery from replication stress. Tolerance to replication stress also depends on the Mec1 kinase, which activates the DNA replication checkpoint in an Mrc1‐dependent manner in response to fork arrest. Although the Mec1 and Rad52 pathways are initiated by the same single‐strand DNA (ssDNA) intermediate, their interplay at stalled forks remains largely unexplored. Here, we show that the replication checkpoint suppresses the formation of Rad52 foci in an Mrc1‐dependent manner and prevents homologous recombination (HR) at chromosome breaks induced by the HO endonuclease. This repression operates at least in part by impeding resection of DNA ends, which is essential to generate 3′ ssDNA tails, the primary substrate of HR. Interestingly, we also observed that the Mec1 pathway does not prevent recombination at stalled forks, presumably because they already contain ssDNA. Taken together, these data indicate that the DNA replication checkpoint suppresses genomic instability in S phase by blocking recombination at chromosome breaks and permitting helpful recombination at stalled forks.     

Rad51 replication fork recruitment is required for DNA damage tolerance

Homologous recombination (HR) is essential for genome integrity. Recombination proteins participate in tolerating DNA lesions that interfere with DNA replication, but can also generate toxic recombination intermediates and genetic instability when they are not properly regulated. Here, we have studied the role of the recombination proteins Rad51 and Rad52 at replication forks and replicative DNA lesions. We show that Rad52 loads Rad51 onto unperturbed replication forks, where they facilitate replication of alkylated DNA by non‐repair functions. The recruitment of Rad52 and Rad51 to chromatin during DNA replication is a prerequisite for the repair of the non‐DSB DNA lesions, presumably single‐stranded DNA gaps, which are generated during the replication of alkylated DNA. We also show that the repair of these lesions requires CDK1 and is not coupled to the fork but rather restricted to G2/M by the replicative checkpoint. We propose a new scenario for HR where Rad52 and Rad51 are recruited to the fork to promote DNA damage tolerance by distinct and cell cycle‐regulated replicative and repair functions.     

Continued DNA Synthesis in Replication Checkpoint Mutants Leads to Fork Collapse

Hydroxyurea (HU) treatment activates the intra-S phase checkpoint proteins Cds1 and Mrc1 to prevent replication fork collapse. We found that prolonged DNA synthesis occurs in cds1Δ and mrc1Δ checkpoint mutants in the presence of HU and continues after release. This is coincident with increased DNA damage measured by phosphorylated histone H2A in whole cells during release. High-resolution live-cell imaging shows that mutants first accumulate extensive replication protein A (RPA) foci, followed by increased Rad52. Both DNA synthesis and RPA accumulation require the MCM helicase. We propose that a replication fork “collapse point” in HU-treated cells describes the point at which accumulated DNA damage and instability at individual forks prevent further replication. After this point, cds1Δ and mrc1Δ forks cannot complete genome replication. These observations establish replication fork collapse as a dynamic process that continues after release from HU block.     

The S‐phase checkpoint: targeting the replication fork

The S‐phase checkpoint is a surveillance mechanism, mediated by the protein kinases Mec1 and Rad53 in the budding yeast Saccharomyces cerevisiae (ATR and Chk2 in human cells, respectively) that responds to DNA damage and replication perturbations by co‐ordinating a global cellular response necessary to maintain genome integrity. A key aspect of this response is the stabilization of DNA replication forks, which is critical for cell survival. A defective checkpoint causes irreversible replication‐fork collapse and leads to genomic instability, a hallmark of cancer cells. Although the precise mechanisms by which Mec1/Rad53 maintain functional replication forks are currently unclear, our knowledge about this checkpoint function has significantly increased during the last years. Focusing mainly on the advances obtained in S. cerevisiae, the present review will summarize our understanding of how the S‐phase checkpoint preserves the integrity of DNA replication forks and discuss the most recent findings on this topic.     

Swi1 prevents replication fork collapse and controls checkpoint kinase Cds1

The replication checkpoint is a dedicated sensor-response system activated by impeded replication forks. It stabilizes stalled forks and arrests division, thereby preserving genome integrity and promoting cell survival. In budding yeast, Tof1 is thought to act as a specific mediator of the replication checkpoint signal that activates the effector kinase Rad53. Here we report studies of fission yeast Swi1, a Tof1-related protein required for a programmed fork-pausing event necessary for mating type switching. Our studies have shown that Swi1 is vital for proficient activation of the Rad53-like checkpoint kinase Cds1. Together they are required to prevent fork collapse in the ribosomal DNA repeats, and they also prevent irreversible fork arrest at a newly identified hydroxyurea pause site. Swi1 also has Cds1-independent functions. Rad22 DNA repair foci form during S phase in swi1 mutants and to a lesser extent in cds1 mutants, indicative of fork collapse. Mus81, a DNA endonuclease required for recovery from collapsed forks, is vital in swi1 but not cds1 mutants. Swi1 is recruited to chromatin during S phase. We propose that Swi1 stabilizes replication forks in a configuration that is recognized by replication checkpoint sensors.     

Replication blocking lesions present a unique substrate for homologous recombination

Homologous recombination (HR) plays a critical role in the restart of blocked replication forks, but how this is achieved remains poorly understood. We show that mutants in the single Rad51 paralog in Caenorhabditis elegans, rfs-1, permit discrimination between HR substrates generated at DNA double-strand breaks (DSBs), or following replication fork collapse from HR substrates assembled at replication fork barriers (RFBs). Unexpectedly, RFS-1 is dispensable for RAD-51 recruitment to meiotic and ionizing radiation (IR)-induced DSBs and following replication fork collapse, yet, is essential for RAD-51 recruitment to RFBs formed by DNA crosslinking agents and other replication blocking lesions. Deletion of rfs-1 also suppresses the accumulation of toxic HR intermediates in him-6; top-3 mutants and accelerates deletion formation at presumed endogenous RFBs formed by poly G/C tracts in the absence of DOG-1. These data suggest that RFS-1 is not a general mediator of HR-dependent DSB repair, but acts specifically to promote HR at RFBs. HR substrates generated at conventional DSBs or following replication fork collapse are therefore intrinsically different from those produced during normal repair of blocked replication forks.     

Limiting amounts of budding yeast Rad53 S-phase checkpoint activity results in increased resistance to DNA alkylation damage

The Saccharomyces cerevisiae protein kinase Rad53 plays a key role in maintaining genomic integrity after DNA damage and is an essential component of the ‘intra-S-phase checkpoint’. In budding yeast, alkylating chemicals, such as methyl methanesulfonate (MMS), or depletion of nucleotides by hydroxyurea (HU) stall DNA replication forks and thus activate Rad53 during S-phase. This stabilizes stalled DNA replication forks and prevents the activation of later origins of DNA replication. Here, we report that a reduction in the level of Rad53 kinase causes cells to behave very differently in response to DNA alkylation or to nucleotide depletion. While cells lacking Rad53 are unable to activate the checkpoint response to HU or MMS, so that they rapidly lose viability, a reduction in Rad53 enhances cell survival only after DNA alkylation. This reduction in the level of Rad53 allows S-phase cells to maintain the stability of DNA replication forks upon MMS treatment, but does not prevent the collapse of forks in HU. Our results may have important implications for cancer therapies, as they suggest that partial impairment of the S-phase checkpoint Rad53/Chk2 kinase provides cells with a growth advantage in the presence of drugs that damage DNA.     

Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Recruitment of the homologous recombination machinery to sites of double‐strand breaks is a cell cycle‐regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B‐type cyclin/CDK1 activity. Induction of the intra‐S‐phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU‐treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra‐S‐phase checkpoint. We propose that B‐type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.     

Fork-Remodeling Helicase Rad5 Preferentially Reverses Replication Forks with Gaps in the Leading Strand

DNA damage bypass pathways promote the replication of damaged DNA when replication forks stall at sites of DNA damage. Template switching is a DNA damage bypass pathway in which fork-reversal helicases convert stalled replication forks into four-way DNA junctions called chicken foot intermediates, which are subsequently extended by replicative DNA polymerases. In yeast, fork-reversal is carried out by the Rad5 helicase using an unknown mechanism. To better understand the mechanism of Rad5 and its specificity for different fork DNA substrates, we used a FRET-based assay to observe fork reversal in real time. We examined the ability of Rad5 to bind and catalyze the reversal of various fork DNA substrates in the presence of short gaps in the leading or lagging strand as well as in the presence or absence of RPA and RNA primers in the lagging strand. We found that Rad5 preferentially reverses fork DNA substrates with short gaps (10 to 30 nt.) in the leading strand. Thus, Rad5 preferentially reverses fork DNA substrates that form chicken foot intermediates with 5′ overhangs that can be extended by replicative DNA polymerases during the subsequent steps of template switching.     

ATM and ATR promote Mre11 dependent restart of collapsed replication forks and prevent accumulation of DNA breaks

Ataxia-telangiectasia mutated (ATM), ataxia-telangiectasia Rad3-related (ATR) and the Mre11/Rad50/Nbs1 complex ensure genome stability in response to DNA damage. However, their essential role in DNA metabolism remains unknown. Here we show that ATM and ATR prevent accumulation of DNA double-strand breaks (DSBs) during chromosomal replication. Replicating chromosomes accumulate DSBs in Xenopus laevis egg extracts depleted of ATM and ATR. Addition of ATM and ATR proteins to depleted extracts prevents DSB accumulation by promoting restart of collapsed replication forks that arise during DNA replication. We show that collapsed forks maintain MCM complex but lose Pol epsilon, and that Pol epsilon reloading requires ATM and ATR. Replication fork restart is abolished in Mre11 depleted extracts and is restored by supplementation with recombinant human Mre11/Rad50/Nbs1 complex. Using a novel fluorescence resonance energy transfer-based technique, we demonstrate that ATM and ATR induce Mre11/Rad50/Nbs1 complex redistribution to restarting forks. This study provides direct biochemical evidence that ATM and ATR prevent accumulation of chromosomal abnormalities by promoting Mre11/Rad50/Nbs1 dependent recovery of collapsed replication forks.     

Identification of SMARCAL1 as a Component of the DNA Damage Response

SMARCAL1 (also known as HARP) is a SWI/SNF family protein with an ATPase activity stimulated by DNA containing both single-stranded and double-stranded regions. Mutations in SMARCAL1 are associated with the disease Schimke immuno-osseous dysplasia, a multisystem autosomal recessive disorder characterized by T cell immunodeficiency, growth inhibition, and renal dysfunction. The cellular function of SMARCAL1, however, is unknown. Here, using Xenopus egg extracts and mass spectrometry, we identify SMARCAL1 as a protein recruited to double-stranded DNA breaks. SMARCAL1 binds to double-stranded breaks and stalled replication forks in both egg extract and human cells, specifically colocalizing with the single-stranded DNA binding factor RPA. In addition, SMARCAL1 interacts physically with RPA independently of DNA. SMARCAL1 is phosphorylated in a caffeine-sensitive manner in response to double-stranded breaks and stalled replication forks. It has been suggested that stalled forks can be stabilized by a mechanism involving caffeine-sensitive kinases, or they collapse and subsequently recruit Rad51 to promote homologous recombination repair. We show that depletion of SMARCAL1 from U2OS cells leads to increased frequency of RAD51 foci upon generation of stalled replication forks, indicating that fork breakdown is more prevalent in the absence of SMARCAL1. We propose that SMARCAL1 is a novel DNA damage-binding protein involved in replication fork stabilization.     

Cooperation of RAD51 and RAD54 in regression of a model replication fork

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the 'template-strand switch' mechanism. This mechanism involves replication fork regression into a Holliday junction ('chicken foot structure'), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion.     

Swi1 and Swi3 are components of a replication fork protection complex in fission yeast

Swi1 is required for programmed pausing of replication forks near the mat1 locus in the fission yeast Schizosaccharomyces pombe. This fork pausing is required to initiate a recombination event that switches mating type. Swi1 is also needed for the replication checkpoint that arrests division in response to fork arrest. How Swi1 accomplishes these tasks is unknown. Here we report that Swi1 copurifies with a 181-amino-acid protein encoded by swi3(+). The Swi1-Swi3 complex is required for survival of fork arrest and for activation of the replication checkpoint kinase Cds1. Association of Swi1 and Swi3 with chromatin during DNA replication correlated with movement of the replication fork. swi1Delta and swi3Delta mutants accumulated Rad22 (Rad52 homolog) DNA repair foci during replication. These foci correlated with the Rad22-dependent appearance of Holliday junction (HJ)-like structures in cells lacking Mus81-Eme1 HJ resolvase. Rhp51 and Rhp54 homologous recombination proteins were not required for viability in swi1Delta or swi3Delta cells, indicating that the HJ-like structures arise from single-strand DNA gaps or rearranged forks instead of broken forks. We propose that Swi1 and Swi3 define a fork protection complex that coordinates leading- and lagging-strand synthesis and stabilizes stalled replication forks.     

PTEN regulates RPA1 and protects DNA replication forks

Tumor suppressor PTEN regulates cellular activities and controls genome stability through multiple mechanisms. In this study, we report that PTEN is necessary for the protection of DNA replication forks against replication stress. We show that deletion of PTEN leads to replication fork collapse and chromosomal instability upon fork stalling following nucleotide depletion induced by hydroxyurea. PTEN is physically associated with replication protein A 1 (RPA1) via the RPA1 C-terminal domain. STORM and iPOND reveal that PTEN is localized at replication sites and promotes RPA1 accumulation on replication forks. PTEN recruits the deubiquitinase OTUB1 to mediate RPA1 deubiquitination. RPA1 deletion confers a phenotype like that observed in PTEN knockout cells with stalling of replication forks. Expression of PTEN and RPA1 shows strong correlation in colorectal cancer. Heterozygous disruption of RPA1 promotes tumorigenesis in mice. These results demonstrate that PTEN is essential for DNA replication fork protection. We propose that RPA1 is a target of PTEN function in fork protection and that PTEN maintains genome stability through regulation of DNA replication.     

Rad22Rad52-dependent repair of ribosomal DNA repeats cleaved by Slx1-Slx4 endonuclease

Slx1 and Slx4 are subunits of a structure-specific DNA endonuclease that is found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, and other eukaryotic species. It is thought to initiate recombination events or process recombination structures that occur during the replication of the tandem repeats of the ribosomal DNA (rDNA) locus. Here, we present evidence that fission yeast Slx1-Slx4 initiates homologous recombination events in the rDNA repeats that are processed by a mechanism that requires Rad22 (Rad52 homologue) but not Rhp51 (Rad51 homologue). Slx1 is required to generate approximately 50% of the spontaneous Rad22 DNA repair foci that occur in cycling cells. Most of these foci colocalize with the nucleolus, which contains the rDNA repeats. The increased fork pausing at the replication fork barriers in the rDNA repeats in a strain that lacks Rqh1 DNA helicase is further increased by expression of a dominant negative form of Slx1. These data suggest that Slx1-Slx4 cleaves paused replication forks in the rDNA, leading to Rad22-dependent homologous recombination that is used to maintain rDNA copy number.     

A dual role of BRCA1 in two distinct homologous recombination mediated repair in response to replication arrest

Homologous recombination (HR) is a major mechanism utilized to repair blockage of DNA replication forks. Here, we report that a sister chromatid exchange (SCE) generated by crossover-associated HR efficiently occurs in response to replication fork stalling before any measurable DNA double-strand breaks (DSBs). Interestingly, SCE produced by replication fork collapse following DNA DSBs creation is specifically suppressed by ATR, a central regulator of the replication checkpoint. BRCA1 depletion leads to decreased RPA2 phosphorylation (RPA2-P) following replication fork stalling but has no obvious effect on RPA2-P following replication fork collapse. Importantly, we found that BRCA1 promotes RAD51 recruitment and SCE induced by replication fork stalling independent of ATR. In contrast, BRCA1 depletion leads to a more profound defect in RAD51 recruitment and SCE induced by replication fork collapse when ATR is depleted. We concluded that BRCA1 plays a dual role in two distinct HR-mediated repair upon replication fork stalling and collapse. Our data established a molecular basis for the observation that defective BRCA1 leads to a high sensitivity to agents that cause replication blocks without being associated with DSBs, and also implicate a novel mechanism by which loss of cell cycle checkpoints promotes BRCA1-associated tumorigenesis via enhancing HR defect resulting from BRCA1 deficiency.     

The ATR pathway: fine-tuning the fork

The proper detection and repair of DNA damage is essential to the maintenance of genomic stability. The genome is particularly vulnerable during DNA replication, when endogenous and exogenous events can hinder replication fork progression. Stalled replication forks can fold into deleterious conformations and are also unstable structures that are prone to collapse or break. These events can lead to inappropriate processing of the DNA, ultimately resulting in genomic instability, chromosomal alterations and cancer. To cope with stalled replication forks, the cell relies on the replication checkpoint to block cell cycle progression, downregulate origin firing, stabilize the fork itself, and restart replication. The ATR (ATM and Rad3-related) kinase and its downstream effector kinase, Chk1, are central regulators of the replication checkpoint. Loss of these checkpoint proteins causes replication fork collapse and chromosomal rearrangements which may ultimately predispose affected individuals to cancer. This review summarizes our current understanding of how the ATR pathway recognizes and stabilizes stalled replication forks.     

Spartan/C1orf124 is important to prevent UV-induced mutagenesis

Uninterrupted replication across damaged DNA is critical to prevent replication fork collapse and resulting double-strand DNA breaks. Rad18-mediated PCNA ubiquitination is a crucial event that triggers a number of downstream pathways important for lesion bypass. Here, we report characterization of Spartan, an evolutionarily conserved protein containing a PCNA-interacting peptide motif, called a PIP box, and a UBZ4 ubiquitin-binding domain. Spartan is a nuclear protein and forms DNA damage-induced foci that colocalize with markers for stalled DNA replication. Focus formation of Spartan requires its PIP-box and the UBZ4 domain and is dependent on Rad18 and the PCNA ubiquitination site, indicating that Spartan is recruited to ubiquitinated PCNA. Spartan depletion results in increased mutagenesis during replication of UV-damaged DNA. Taken together, our data suggest that Spartan is recruited to sites of stalled replication via ubiquitinated PCNA and plays an important role to prevent mutations associated with replication of damaged DNA.