DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.     

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.     

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.     

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.     

Unwinding of the nascent lagging strand by Rep and PriA enables the direct restart of stalled replication forks

During origin-independent replisome assembly, the replication restart protein PriC prefers to load the replication fork helicase, DnaB, to stalled replication forks where there is a gap in the nascent leading strand. However, this activity can be obstructed if the 5'-end of the nascent lagging strand is near the template branch point. Here we provide biochemical evidence that the helicase activities of Rep and PriA function to unwind the nascent lagging strand DNA at such stalled replication forks. PriC then loads the replicative helicase, DnaB, onto the newly generated, single-stranded template for the purposes of replisome assembly and duplex unwinding ahead of the replication fork. Direct rescue of replication forks by the Rep-PriC and PriA-PriC pathways in this manner may contribute to genomic stability by avoiding the potential dangers of fork breakage inherent to recombination-dependent restart pathways.     

WRNIP1 protects stalled forks from degradation and promotes fork restart after replication stress

Accurate handling of stalled replication forks is crucial for the maintenance of genome stability. RAD51 defends stalled replication forks from nucleolytic attack, which otherwise can threaten genome stability. However, the identity of other factors that can collaborate with RAD51 in this task is poorly elucidated. Here, we establish that human Werner helicase interacting protein 1 (WRNIP1) is localized to stalled replication forks and cooperates with RAD51 to safeguard fork integrity. We show that WRNIP1 is directly involved in preventing uncontrolled MRE11‐mediated degradation of stalled replication forks by promoting RAD51 stabilization on ssDNA. We further demonstrate that replication fork protection does not require the ATPase activity of WRNIP1 that is however essential to achieve the recovery of perturbed replication forks. Loss of WRNIP1 or its catalytic activity causes extensive DNA damage and chromosomal aberrations. Intriguingly, downregulation of the anti‐recombinase FBH1 can compensate for loss of WRNIP1 activity, since it attenuates replication fork degradation and chromosomal aberrations in WRNIP1‐deficient cells. Therefore, these findings unveil a unique role for WRNIP1 as a replication fork‐protective factor in maintaining genome stability.     

Human DNA polymerase eta promotes DNA synthesis from strand invasion intermediates of homologous recombination

Stalled replication forks pose a serious threat to genome integrity. To overcome the catastrophic consequences associated with fork demise, translesion synthesis (TLS) polymerases such as poleta promote DNA synthesis past lesions. Alternatively, a stalled fork may collapse and undergo repair by homologous recombination. By using fractionated cell extracts and purified recombinant proteins, we show that poleta extends DNA synthesis from D loop recombination intermediates in which an invading strand serves as the primer. Extracts from XP-V cells, which are defective in poleta, exhibit severely reduced D loop extension activity. The D loop extension activity of poleta is unusual, as this reaction cannot be promoted by the replicative DNA polymerase delta or by other TLS polymerases such as poliota. Moreover, we find that poleta interacts with RAD51 recombinase and RAD51 stimulates poleta-mediated D loop extension. Our results indicate a dual function for poleta at stalled replication forks: the promotion of translesion synthesis and the reinitiation of DNA synthesis by homologous recombination repair.     

Maintaining replication fork integrity in UV-irradiated Escherichia coli cells

In dividing cells, the stalling of replication fork complexes by impediments to DNA unwinding or by template imperfections that block synthesis by the polymerase subunits is a serious threat to genomic integrity and cell viability. What happens to stalled forks depends on the nature of the offending obstacle. In UV-irradiated Escherichia coli cells DNA synthesis is delayed for a considerable period, during which forks undergo extensive processing before replication can resume. Thus, restart depends on factors needed to load the replicative helicase, indicating that the replisome may have dissociated. It also requires the RecFOR proteins, which are known to load RecA recombinase on single-stranded DNA, implying that template strands are exposed. To gain a further understanding of how UV irradiation affects replication and how replication resumes after a block, we used fluorescence microscopy and BrdU or radioisotope labelling to examine chromosome replication and cell cycle progression. Our studies confirm that RecFOR promote efficient reactivation of stalled forks and demonstrate that they are also needed for productive replication initiated at the origin, or triggered elsewhere by damage to the DNA. Although delayed, all modes of replication do recover in the absence of these proteins, but nascent DNA strands are degraded more extensively by RecJ exonuclease. However, these strands are also degraded in the presence of RecFOR when restart is blocked by other means, indicating that RecA loading is not sufficient to stabilise and protect the fork. This is consistent with the idea that RecA actively promotes restart. Thus, in contrast to eukaryotic cells, there may be no factor in bacterial cells acting specifically to stabilise stalled forks. Instead, nascent strands may be protected by the simple expedient of promoting restart. We also report that the efficiency of fork reactivation is not affected in polB mutants.     

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.     

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.     

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.     

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.     

FANCD2-Controlled Chromatin Access of the Fanconi-Associated Nuclease FAN1 Is Crucial for the Recovery of Stalled Replication Forks

Fanconi anemia (FA) is a cancer predisposition syndrome characterized by cellular hypersensitivity to DNA interstrand cross-links (ICLs). Within the FA pathway, an upstream core complex monoubiquitinates and recruits the FANCD2 protein to ICLs on chromatin. Ensuing DNA repair involves the Fanconi-associated nuclease 1 (FAN1), which interacts selectively with monoubiquitinated FANCD2 (FANCD2^Ub) at ICLs. Importantly, FANCD2 has additional independent functions: it binds chromatin and coordinates the restart of aphidicolin (APH)-stalled replication forks in concert with the BLM helicase, while protecting forks from nucleolytic degradation by MRE11. We identified FAN1 as a new crucial replication fork recovery factor. FAN1 joins the BLM-FANCD2 complex following APH-mediated fork stalling in a manner dependent on MRE11 and FANCD2, followed by FAN1 nuclease-mediated fork restart. Surprisingly, APH-induced activation and chromatin recruitment of FAN1 occur independently of the FA core complex or the FAN1 UBZ domain, indicating that the FANCD2^Ub isoform is dispensable for functional FANCD2-FAN1 cross talk during stalled fork recovery. In the absence of FANCD2, MRE11 exonuclease-promoted access of FAN1 to stalled forks results in severe FAN1-mediated nucleolytic degradation of nascent DNA strands. Thus, FAN1 nuclease activity at stalled replication forks requires tight regulation: too little inhibits fork restart, whereas too much causes fork degradation.     

Polyubiquitinated PCNA Recruits the ZRANB3 Translocase to Maintain Genomic Integrity after Replication Stress

Completion of DNA replication after replication stress depends on PCNA, which undergoes monoubiquitination to stimulate direct bypass of DNA lesions by specialized DNA polymerases or is polyubiquitinated to promote recombination-dependent DNA synthesis across DNA lesions by template switching mechanisms. Here we report that the ZRANB3 translocase, a SNF2 family member related to the SIOD disorder SMARCAL1 protein, is recruited by polyubiquitinated PCNA to promote fork restart following replication arrest. ZRANB3 depletion in mammalian cells results in an increased frequency of sister chromatid exchange and DNA damage sensitivity after treatment with agents that cause replication stress. Using in?vitro biochemical assays, we show that recombinant ZRANB3 remodels DNA?structures mimicking stalled replication forks and disassembles recombination intermediates. We therefore propose that ZRANB3 maintains genomic stability at stalled or collapsed replication forks by facilitating fork restart and limiting inappropriate recombination that could occur during template switching events.     

EEPD1 Rescues Stressed Replication Forks and Maintains Genome Stability by Promoting End Resection and Homologous Recombination Repair

Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5’ end resection near the fork junction, which permits 3’ single strand invasion of a homologous template for fork restart. This 5’ end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5’ DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5’ overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.     

Building up and breaking down: mechanisms controlling recombination during replication

The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.     

Replisome fate upon encountering a leading strand block and clearance from DNA by recombination proteins

Replication forks that collapse upon encountering a leading strand lesion are reactivated by a recombinative repair process called replication restart. Using rolling circle DNA substrates to model replication forks, we examine the fate of the helicase and both DNA polymerases when the leading strand polymerase is blocked. We find that the helicase continues over 0.5 kb but less than 3 kb and that the lagging strand DNA polymerase remains active despite its connection to a stalled leading strand enzyme. Furthermore, the blocked leading strand polymerase remains stably bound to the replication fork, implying that it must be dismantled from DNA in order for replication restart to initiate. Genetic studies have identified at least four gene products required for replication restart, RecF, RecO, RecR, and RecA. We find here that these proteins displace a stalled polymerase at a DNA template lesion. Implications of these results for replication fork collapse and recovery are discussed.     

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.     

The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing

When replication forks stall at damaged bases or upon nucleotide depletion, the intra-S phase checkpoint ensures they are stabilized and can restart. In intra-S checkpoint-deficient budding yeast, stalling forks collapse, and ～10% form pathogenic chicken foot structures, contributing to incomplete replication and cell death (Lopes et al., 2001; Sogo et al., 2002; Tercero and Diffley, 2001). Using fission yeast, we report that the Cds1(Chk2) effector kinase targets Dna2 on S220 to regulate, both in vivo and in vitro, Dna2 association with stalled replication forks in chromatin. We demonstrate that Dna2-S220 phosphorylation and the nuclease activity of Dna2 are required to prevent fork reversal. Consistent with this, Dna2 can efficiently cleave obligate precursors of fork regression-regressed leading or lagging strands-on model replication forks. We propose that Dna2 cleavage of regressed nascent strands prevents fork reversal and thus stabilizes stalled forks to maintain genome stability during replication stress.     

Cockayne syndrome group B protein regulates fork restart,fork progression and MRE11-dependent fork degradation in BRCA1/2-deficient cells

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.