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.     

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.     

Chromatin assembly controls replication fork stability

During DNA replication, the advance of replication forks is tightly connected with chromatin assembly, a process that can be impaired by the partial depletion of histone H4 leading to recombinogenic DNA damage. Here, we show that the partial depletion of H4 is rapidly followed by the collapse of unperturbed and stalled replication forks, even though the S‐phase checkpoints remain functional. This collapse is characterized by a reduction in the amount of replication intermediates, but an increase in single Ys relative to bubbles, defects in the integrity of the replisome and an accumulation of DNA double‐strand breaks. This collapse is also associated with an accumulation of Rad52‐dependent X‐shaped molecules. Consistently, a Rad52‐dependent—although Rad51‐independent—mechanism is able to rescue these broken replication forks. Our findings reveal that correct nucleosome deposition is required for replication fork stability, and provide molecular evidence for homologous recombination as an efficient mechanism of replication fork restart.     

Claspin and Chk1 regulate replication fork stability by different mechanisms

The checkpoint mediator protein Claspin facilitates the phosphorylation and activation of Chk1 by ATR and thus is required for efficient DNA replication. However, the physical association of Claspin homologues with replication factors and forks suggests that it might have additional functions in controlling DNA replication. DNA combing was used to examine the functions of Chk1 and Claspin at individual forks and to determine whether Claspin functions independently of Chk1. We find that Claspin, like Chk1, regulates fork stability and density in unperturbed cells. As expected, Chk1 regulates origin firing predominantly by controlling Cdk2-Cdc25 function. By contrast, Claspin functions independently of the Cdc25-Cdk2 pathway in mammalian cells. The findings support a model in which Claspin plays a role regulating replication fork stability that is independent of its function in mediating Chk1 phosphorylation.     

Separation of intra-S checkpoint protein contributions to DNA replication fork protection and genomic stability in normal human fibroblasts

The ATR-dependent intra-S checkpoint protects DNA replication forks undergoing replication stress. The checkpoint is enforced by ATR-dependent phosphorylation of CHK1, which is mediated by the TIMELESS-TIPIN complex and CLASPIN. Although loss of checkpoint proteins is associated with spontaneous chromosomal instability, few studies have examined the contribution of these proteins to unchallenged DNA metabolism in human cells that have not undergone carcinogenesis or crisis. Furthermore, the TIMELESS-TIPIN complex and CLASPIN may promote replication fork protection independently of CHK1 activation. Normal human fibroblasts (NHF) were depleted of ATR, CHK1, TIMELESS, TIPIN or CLASPIN and chromosomal aberrations, DNA synthesis, activation of the DNA damage response (DDR) and clonogenic survival were evaluated. This work demonstrates in NHF lines from two individuals that ATR and CHK1 promote chromosomal stability by different mechanisms that depletion of CHK1 produces phenotypes that resemble more closely the depletion of TIPIN or CLASPIN than the depletion of ATR, and that TIMELESS has a distinct contribution to suppression of chromosomal instability that is independent of its heterodimeric partner, TIPIN. Therefore, ATR, CHK1, TIMELESS-TIPIN and CLASPIN have functions for preservation of intrinsic chromosomal stability that are separate from their cooperation for activation of the intra-S checkpoint response to experimentally induced replication stress. These data reveal a complex and coordinated program of genome maintenance enforced by proteins known for their intra-S checkpoint function.     

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.     

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.     

RMI1 promotes DNA replication fork progression and recovery from replication fork stress

RMI1 is a member of an evolutionarily conserved complex composed of BLM and topoisomerase IIIα (TopoIIIα). This complex exhibits strand passage activity in vitro, which is likely important for DNA repair and DNA replication in vivo. The inactivation of RMI1 causes genome instability, including elevated levels of sister chromatid exchange and accelerated tumorigenesis. Using molecular combing to analyze DNA replication at the single-molecule level, we show that RMI1 is required to promote normal replication fork progression. The fork progression defect in RMI1-depleted cells is alleviated in cells lacking BLM, indicating that RMI1 functions downstream of BLM in promoting replication elongation. RMI1 localizes to subnuclear foci with BLM and TopoIIIα in response to replication stress. The proper localization of the complex requires a BLM-TopoIIIα-RMI1 interaction and is essential for RMI1 to promote recovery from replication stress. These findings reveal direct roles of RMI1 in DNA replication and the replication stress response, which could explain the molecular basis for its involvement in suppressing sister chromatid exchange and tumorigenesis.     

The role of SMARCAL1 in replication fork stability and telomere maintenance

SMARCAL1 (SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of Chromatin, Subfamily A-Like 1), also known as HARP, is an ATP-dependent annealing helicase that stabilizes replication forks during DNA damage. Mutations in this gene are the cause of Schimke immune-osseous dysplasia (SIOD), an autosomal recessive disorder characterized by T-cell immunodeficiency and growth dysfunctions. In this review, we summarize the main roles of SMARCAL1 in DNA repair, telomere maintenance and replication fork stability in response to DNA replication stress.     

DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.     

Mechanistically distinct roles for Sgs1p in checkpoint activation and replication fork maintenance

The RecQ helicase Sgs1p forms a complex with the type 1 DNA topoisomerase Top3p that resolves double Holliday junctions resulting from Rad51-mediated exchange. We find, however, that Sgs1p functions independently of both Top3p and Rad51p to stimulate the checkpoint kinase Rad53p when replication forks stall due to dNTP depletion on hydroxyurea. Checkpoint activation does not require Sgs1p function as a helicase, and correlates with its ability to bind the Rad53p kinase FHA1 motif directly. On the other hand, Sgs1p's helicase activity is required together with Top3p and the strand-exchange factor Rad51p, to help stabilise DNA polymerase epsilon at stalled replication forks. In this function, the Sgs1p/Top3p complex acts in parallel to the Claspin-related adaptor, Mrc1p, although the sgs1 and mrc1 mutations are epistatic for Rad53p activation. We thus identify two distinct pathways through which Sgs1p contributes to genomic integrity: checkpoint kinase activation requires Sgs1p as a noncatalytic Rad53p-binding site, while the combined Top3p/Sgs1p resolvase activity contributes to replisome stability and recovery from arrested replication forks.     

Chromosome replication: from ORC to fork

A report on the 2001 Eukaryotic DNA Replication meeting, Cold Spring Harbor Laboratory, New York, 5-9 September 2001.     

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.     

The replication fork trap and termination of chromosome replication

Bacteria that have a circular chromosome with a bidirectional DNA replication origin are thought to utilize a ‘replication fork trap’ to control termination of replication. The fork trap is an arrangement of replication pause sites that ensures that the two replication forks fuse within the terminus region of the chromosome, approximately opposite the origin on the circular map. However, the biological significance of the replication fork trap has been mysterious, as its inactivation has no obvious consequence. Here we review the research that led to the replication fork trap theory, and we aim to integrate several recent findings that contribute towards an understanding of the physiological roles of the replication fork trap. Likely roles include the prevention of over‐replication, and the optimization of post‐replicative mechanisms of chromosome segregation, such as that involving FtsK in Escherichia coli.     

Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.     

Mechanisms of replication fork protection: a safeguard for genome stability

During S-phase, the genome is extremely vulnerable and the progression of replication forks is often threatened by exogenous and endogenous challenges. When replication fork progression is halted, the intra S-phase checkpoint is activated to promote structural stability of stalled forks, preventing the dissociation of replisome components. This ensures the rapid resumption of replication following DNA repair. Failure in protecting and/or restarting the stalled forks contributes to alterations of the genome. Several human genetic diseases coupled to an increased cancer predisposition are caused by mutations in genes involved in safeguarding genome integrity during DNA replication. Both the ATR (ataxia telangiectasia and Rad3-related protein) kinase and the Replication pausing complex (RPC) components Tipin, Tim1 and Claspin play key roles in activating the intra S-phase checkpoint and in stabilizing the stalled replication forks. Here, we discuss the specific contribution of these factors in preserving fork structure and ensuring accurate completion of DNA replication.