ATR and ATM differently regulate WRN to prevent DSBs at stalled replication forks and promote replication fork recovery

Accurate response to replication arrest is crucial to preserve genome stability and requires both the ATR and ATM functions. The Werner syndrome protein (WRN) is implicated in the recovery of stalled replication forks, and although an ATR/ATM‐dependent phosphorylation of WRN was observed after replication arrest, the function of such modifications during the response to perturbed replication is not yet appreciated. Here, we report that WRN is directly phosphorylated by ATR at multiple C‐terminal S/TQ residues. Suppression of ATR‐mediated phosphorylation of WRN prevents proper accumulation of WRN in nuclear foci, co‐localisation with RPA and causes breakage of stalled forks. On the other hand, inhibition of ATM kinase activity or expression of an ATM‐unphosphorylable WRN allele leads to retention of WRN in nuclear foci and impaired recruitment of RAD51 recombinase resulting in reduced viability after fork collapse. Altogether, our findings indicate that ATR and ATM promote recovery from perturbed replication by differently regulating WRN at defined moments of the response to replication fork arrest.     

Oligomerization of DNA replication regulatory protein RADX is essential to maintain replication fork stability

Genome integrity requires complete and accurate DNA replication once per cell division cycle. Replication stress poses obstacles to this process that must be overcome to prevent replication fork collapse. An important regulator of replication fork stability is the RAD51 protein, which promotes replication fork reversal and protects nascent DNA strands from nuclease-mediated degradation. Many regulatory proteins control these RAD51 activities, including RADX, which binds both ssDNA and RAD51 at replication forks to ensure that fork reversal is confined to stalled forks. Many ssDNA-binding proteins function as hetero- or homo-oligomers. In this study, we addressed whether this is also the case for RADX. Using biochemical and genetic approaches, we found that RADX acts as a homo-oligomer to control replication fork stability. RADX oligomerizes using at least two different interaction surfaces, including one mapped to a C-terminal region. We demonstrate that mutations in this region prevent oligomerization and prevent RADX function in cells, and that addition of a heterologous dimerization domain to the oligomerization mutants restored their ability to regulate replication. Taken together, our results demonstrate that like many ssDNA-binding proteins, oligomerization is essential for RADX-mediated regulation of genome stability.     

Lamin A/C recruits ssDNA protective proteins RPA and RAD51 to stalled replication forks to maintain fork stability

Lamin A/C provides a nuclear scaffold for compartmentalization of genome function that is important for genome integrity. Lamin A/C dysfunction is associated with cancer, aging, and degenerative diseases. The mechanisms whereby lamin A/C regulates genome stability remain poorly understood. We demonstrate a crucial role for lamin A/C in DNA replication. Lamin A/C binds to nascent DNA, especially during replication stress (RS), ensuring the recruitment of replication fork protective factors RPA and RAD51. These ssDNA-binding proteins, considered the first and second responders to RS respectively, function in the stabilization, remodeling, and repair of the stalled fork to ensure proper restart and genome stability. Reduced recruitment of RPA and RAD51 upon lamin A/C depletion elicits replication fork instability (RFI) characterized by MRE11 nuclease–mediated degradation of nascent DNA, RS-induced DNA damage, and sensitivity to replication inhibitors. Importantly, unlike homologous recombination–deficient cells, RFI in lamin A/C-depleted cells is not linked to replication fork reversal. Thus, the point of entry of nucleases is not the reversed fork but regions of ssDNA generated during RS that are not protected by RPA and RAD51. Consistently, RFI in lamin A/C-depleted cells is rescued by exogenous overexpression of RPA or RAD51. These data unveil involvement of structural nuclear proteins in the protection of ssDNA from nucleases during RS by promoting recruitment of RPA and RAD51 to stalled forks. Supporting this model, we show physical interaction between RPA and lamin A/C. We suggest that RS is a major source of genomic instability in laminopathies and lamin A/C-deficient tumors.     

Mammalian RAD51 paralogs protect nascent DNA at stalled forks and mediate replication restart

Mammalian RAD51 paralogs are implicated in the repair of collapsed replication forks by homologous recombination. However, their physiological roles in replication fork maintenance prior to fork collapse remain obscure. Here, we report on the role of RAD51 paralogs in short-term replicative stress devoid of DSBs. We show that RAD51 paralogs localize to nascent DNA and common fragile sites upon replication fork stalling. Strikingly, RAD51 paralogs deficient cells exhibit elevated levels of 53BP1 nuclear bodies and increased DSB formation, the latter being attributed to extensive degradation of nascent DNA at stalled forks. RAD51C and XRCC3 promote the restart of stalled replication in an ATP hydrolysis dependent manner by disengaging RAD51 and other RAD51 paralogs from the halted forks. Notably, we find that Fanconi anemia (FA)-like disorder and breast and ovarian cancer patient derived mutations of RAD51C fails to protect replication fork, exhibit under-replicated genomic regions and elevated micro-nucleation. Taken together, RAD51 paralogs prevent degradation of stalled forks and promote the restart of halted replication to avoid replication fork collapse, thereby maintaining genomic integrity and suppressing tumorigenesis.     

真核细胞染色体DNA复制起始及复制叉稳定性维持的机制

在真核生物中,DNA复制在染色体上特定的多位点起始．当细胞处在晚M及G1期,多个复制起始蛋白依次结合到DNA复制源,组装形成复制前复合体．pre．RC在Gl-S的转折期得到激活,随后,多个直接参与DNA复制又形成的蛋白结合到DNA复制源,启动DNA的复制,形成两个双向的DNA复制又．在染色体上,移动的DNA复制又经常会碰到复制障碍（二级DNA结构、一些蛋白的结合位点、损伤的碱基等）而暂停下来,此时,需要细胞周期检验点的调控来稳定复制叉,否则,会导致复制又垮塌及基因组不稳定．本文就真核细胞染色体DNA复制起始的机制,以及复制又稳定性的维持机制进行简要综述．     

The MMS22L–TONSL heterodimer directly promotes RAD51‐dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double‐strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L–TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L–TONSL associates with replication protein A (RPA)‐coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR‐mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L–TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51‐ssDNA nucleoprotein filament formation and RAD51‐dependent strand exchange activity in vitro. Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L–TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR‐mediated restart in vivo.     

Stalled replication forks: Making ends meet for recognition and stabilization

In bacteria, PriA protein, a conserved DEXH‐type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA‐binding property allows it to recognize and stabilize stalled forks and the structures derived from them. Cells must cope with fork stalls caused by various replication stresses to complete replication of the entire genome. Failure of the stalled fork stabilization process and eventual restart could lead to various forms of genomic instability. The low viability of priA null cells indicates a frequent occurrence of fork stall during normal growth that needs to be properly processed. PriA specifically recognizes the 3′‐terminus of the nascent leading strand or the invading strand in a displacement (D)‐loop by the three‐prime terminus binding pocket (TT‐pocket) present in its unique DNA binding domain. Elucidation of the structural basis for recognition of arrested forks by PriA should provide useful insight into how stalled forks are recognized in eukaryotes.     

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.     

Double-strand break repair-independent role for BRCA2 in blocking stalled replication fork degradation by MRE11

Breast cancer suppressor BRCA2 is critical for maintenance of genomic integrity and resistance to agents that damage DNA or collapse replication forks, presumably through homology-directed repair of double-strand breaks (HDR). Using single-molecule DNA fiber analysis, we show here that nascent replication tracts created before fork stalling with hydroxyurea are degraded in the absence of BRCA2 but are stable in wild-type cells. BRCA2 mutational analysis reveals that a conserved C-terminal site involved in stabilizing RAD51 filaments, but not in loading RAD51 onto DNA, is essential for this fork protection but dispensable for HDR. RAD51 filament disruption in wild-type cells phenocopies BRCA2 deficiency. BRCA2 prevents chromosomal aberrations on replication stalling, which are alleviated by inhibition of MRE11, the nuclease responsible for this form of fork instability. Thus, BRCA2 prevents rather than repairs nucleolytic lesions at stalled replication forks to maintain genomic integrity and hence likely suppresses tumorigenesis through this replication-specific function.     

Mechanisms for stalled replication fork stabilization: new targets for synthetic lethality strategies in cancer treatments

Timely and faithful duplication of the entire genome depends on completion of replication. Replication forks frequently encounter obstacles that may cause genotoxic fork stalling. Nevertheless, failure to complete replication rarely occurs under normal conditions, which is attributed to an intricate network of proteins that serves to stabilize, repair and restart stalled forks. Indeed, many of the components in this network are encoded by tumour suppressor genes, and their loss of function by mutation or deletion generates genomic instability, a hallmark of cancer. Paradoxically, the same fork‐protective network also confers resistance of cancer cells to chemotherapeutic drugs that induce high‐level replication stress. Here, we review the mechanisms and major pathways rescuing stalled replication forks, with a focus on fork stabilization preventing fork collapse. A coherent understanding of how cells protect their replication forks will not only provide insight into how cells maintain genome stability, but also unravel potential therapeutic targets for cancers refractory to conventional chemotherapies.     

FANCM regulates DNA chain elongation and is stabilized by S‐phase checkpoint signalling

FANCM binds and remodels replication fork structures in vitro. We report that in vivo, FANCM controls DNA chain elongation in an ATPase‐dependent manner. In the presence of replication inhibitors that do not damage DNA, FANCM counteracts fork movement, possibly by remodelling fork structures. Conversely, through damaged DNA, FANCM promotes replication and recovers stalled forks. Hence, the impact of FANCM on fork progression depends on the underlying hindrance. We further report that signalling through the checkpoint effector kinase Chk1 prevents FANCM from degradation by the proteasome after exposure to DNA damage. FANCM also acts in a feedback loop to stabilize Chk1. We propose that FANCM is a ringmaster in the response to replication stress by physically altering replication fork structures and by providing a tight link to S‐phase checkpoint signalling.     

RAD51 Mutants Cause Replication Defects and Chromosomal Instability

RAD51 is important for restarting stalled replication forks and for repairing DNA double-strand breaks (DSBs) through a pathway called homology-directed repair (HDR). However, analysis of the consequences of specific RAD51 mutants has been difficult since they are toxic. Here we report on the dominant effects of two human RAD51 mutants defective for ATP binding (K133A) or ATP hydrolysis (K133R) expressed in mouse embryonic stem (ES) cells that also expressed normal mouse RAD51 from the other chromosome. These cells were defective for restarting stalled replication forks and repairing breaks. They were also hypersensitive to camptothecin, a genotoxin that generates breaks specifically at the replication fork. In addition, these cells exhibited a wide range of structural chromosomal changes that included multiple breakpoints within the same chromosome. Thus, ATP binding and hydrolysis are essential for chromosomal maintenance. Fusion of RAD51 to a fluorescent tag (enhanced green fluorescent protein [eGFP]) allowed visualization of these proteins at sites of replication and repair. We found very low levels of mutant protein present at these sites compared to normal protein, suggesting that low levels of mutant protein were sufficient for disruption of RAD51 activity and generation of chromosomal rearrangements.     

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.     

The emerging determinants of replication fork stability

A universal response to replication stress is replication fork reversal, where the nascent complementary DNA strands are annealed to form a protective four-way junction allowing forks to avert DNA damage while replication stress is resolved. However, reversed forks are in turn susceptible to nucleolytic digestion of the regressed nascent DNA arms and rely on dedicated mechanisms to protect their integrity. The most well studied fork protection mechanism involves the BRCA pathway and its ability to catalyze RAD51 nucleofilament formation on the reversed arms of stalled replication forks. Importantly, the inability to prevent the degradation of reversed forks has emerged as a hallmark of BRCA deficiency and underlies genome instability and chemosensitivity in BRCA-deficient cells. In the past decade, multiple factors underlying fork stability have been discovered. These factors either cooperate with the BRCA pathway, operate independently from it to augment fork stability in its absence, or act as enablers of fork degradation. In this review, we examine these novel determinants of fork stability, explore the emergent conceptual underpinnings underlying fork protection, as well as the impact of fork protection on cellular viability and cancer therapy.     

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.     

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.     

Fork regression is an active helicase‐driven pathway in bacteriophage T4

Reactivation of stalled replication forks requires specialized mechanisms that can recognize the fork structure and promote downstream processing events. Fork regression has been implicated in several models of fork reactivation as a crucial processing step that supports repair. However, it has also been suggested that regressed forks represent pathological structures rather than physiological intermediates of repair. To investigate the biological role of fork regression in bacteriophage T4, we tested several mechanistic models of regression: strand exchange‐mediated extrusion, topology‐driven fork reversal and helicase‐mediated extrusion. Here, we report that UvsW, a T4 branch‐specific helicase, is necessary for the accumulation of regressed forks in vivo, and that UvsW‐catalysed regression is the dominant mechanism of origin‐fork processing that contributes to double‐strand end formation. We also show that UvsW resolves purified fork intermediates in vitro by fork regression. Regression is therefore part of an active, UvsW‐driven pathway of fork processing in bacteriophage T4.     

Rad9/53BP1 protects stalled replication forks from degradation in Mec1/ATR‐defective cells

Nucleolytic processing by nucleases can be a relevant mechanism to allow repair/restart of stalled replication forks. However, nuclease action needs to be controlled to prevent overprocessing of damaged replication forks that can be detrimental to genome stability. The checkpoint protein Rad9/53BP1 is known to limit nucleolytic degradation (resection) of DNA double‐strand breaks (DSBs) in both yeast and mammals. Here, we show that loss of the inhibition that Rad9 exerts on resection exacerbates the sensitivity to replication stress of Mec1/ATR‐defective yeast cells by exposing stalled replication forks to Dna2‐dependent degradation. This Rad9 protective function is independent of checkpoint activation and relies mainly on Rad9‐Dpb11 interaction. We propose that Rad9/53BP1 supports cell viability by protecting stalled replication forks from extensive resection when the intra‐S checkpoint is not fully functional.     

Replication fork blockage by RTS1 at an ectopic site promotes recombination in fission yeast

Homologous recombination is believed to play important roles in processing stalled/blocked replication forks in eukaryotes. In accordance with this, recombination is induced by replication fork barriers (RFBs) within the rDNA locus. However, the rDNA locus is a specialised region of the genome, and therefore the action of recombinases at its RFBs may be atypical. We show here for the first time that direct repeat recombination, dependent on Rad22 and Rhp51, is induced by replication fork blockage at a site-specific RFB (RTS1) within a 'typical' genomic locus in fission yeast. Importantly, when the RFB is positioned between the direct repeat, conservative gene conversion events predominate over deletion events. This is consistent with recombination occurring without breakage of the blocked fork. In the absence of the RecQ family DNA helicase Rqh1, deletion events increase dramatically, which correlates with the detection of one-sided DNA double-strand breaks at or near RTS1. These data indicate that Rqh1 acts to prevent blocked replication forks from collapsing and thereby inducing deletion events.