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.     

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.     

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.     

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.     

The homologous recombination component EEPD1 is required for genome stability in response to developmental stress of vertebrate embryogenesis

Stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR), initiated by nuclease cleavage of branched structures at stalled forks. We previously reported that the 5′ nuclease EEPD1 is recruited to stressed replication forks, where it plays critical early roles in HR initiation by promoting fork cleavage and end resection. HR repair of stressed replication forks prevents their repair by non-homologous end-joining (NHEJ), which would cause genome instability. Rapid cell division during vertebrate embryonic development generates enormous pressure to maintain replication speed and accuracy. To determine the role of EEPD1 in maintaining replication fork integrity and genome stability during rapid cell division in embryonic development, we assessed the role of EEPD1 during zebrafish embryogenesis. We show here that when EEPD1 is depleted, zebrafish embryos fail to develop normally and have a marked increase in death rate. Zebrafish embryos depleted of EEPD1 are far more sensitive to replication stress caused by nucleotide depletion. We hypothesized that the HR defect with EEPD1 depletion would shift repair of stressed replication forks to unopposed NHEJ, causing chromosome abnormalities. Consistent with this, EEPD1 depletion results in nuclear defects including anaphase bridges and micronuclei in stressed zebrafish embryos, similar to BRCA1 deficiency. These results demonstrate that the newly characterized HR protein EEPD1 maintains genome stability during embryonic replication stress. These data also imply that the rapid cell cycle transit seen during embryonic development produces replication stress that requires HR to resolve.     

14-3-3 Proteins regulate exonuclease 1-dependent processing of stalled replication forks

Replication fork integrity, which is essential for the maintenance of genome stability, is monitored by checkpoint-mediated phosphorylation events. 14-3-3 proteins are able to bind phosphorylated proteins and were shown to play an undefined role under DNA replication stress. Exonuclease 1 (Exo1) processes stalled replication forks in checkpoint-defective yeast cells. We now identify 14-3-3 proteins as in vivo interaction partners of Exo1, both in yeast and mammalian cells. Yeast 14-3-3-deficient cells fail to induce Mec1-dependent Exo1 hyperphosphorylation and accumulate Exo1-dependent ssDNA gaps at stalled forks, as revealed by electron microscopy. This leads to persistent checkpoint activation and exacerbated recovery defects. Moreover, using DNA bi-dimensional electrophoresis, we show that 14-3-3 proteins promote fork progression under limiting nucleotide concentrations. We propose that 14-3-3 proteins assist in controlling the phosphorylation status of Exo1 and additional unknown targets, promoting fork progression, stability, and restart in response to DNA replication stress.     

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.     

SUMOylation mediates CtIP’s functions in DNA end resection and replication fork protection

Double-strand breaks and stalled replication forks are a significant threat to genomic stability that can lead to chromosomal rearrangements or cell death. The protein CtIP promotes DNA end resection, an early step in homologous recombination repair, and has been found to protect perturbed forks from excessive nucleolytic degradation. However, it remains unknown how CtIP’s function in fork protection is regulated. Here, we show that CtIP recruitment to sites of DNA damage and replication stress is impaired upon global inhibition of SUMOylation. We demonstrate that CtIP is a target for modification by SUMO-2 and that this occurs constitutively during S phase. The modification is dependent on the activities of cyclin-dependent kinases and the PI-3-kinase-related kinase ATR on CtIP’s carboxyl-terminal region, an interaction with the replication factor PCNA, and the E3 SUMO ligase PIAS4. We also identify residue K578 as a key residue that contributes to CtIP SUMOylation. Functionally, a CtIP mutant where K578 is substituted with a non-SUMOylatable arginine residue is defective in promoting DNA end resection, homologous recombination, and in protecting stalled replication forks from excessive nucleolytic degradation. Our results shed further light on the tightly coordinated regulation of CtIP by SUMOylation in the maintenance of genome stability.     

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.     

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．     

TRAIP is a PCNA-binding ubiquitin ligase that protects genome stability after replication stress

Cellular genomes are highly vulnerable to perturbations to chromosomal DNA replication. Proliferating cell nuclear antigen (PCNA), the processivity factor for DNA replication, plays a central role as a platform for recruitment of genome surveillance and DNA repair factors to replication forks, allowing cells to mitigate the threats to genome stability posed by replication stress. We identify the E3 ubiquitin ligase TRAIP as a new factor at active and stressed replication forks that directly interacts with PCNA via a conserved PCNA-interacting peptide (PIP) box motif. We show that TRAIP promotes ATR-dependent checkpoint signaling in human cells by facilitating the generation of RPA-bound single-stranded DNA regions upon replication stress in a manner that critically requires its E3 ligase activity and is potentiated by the PIP box. Consequently, loss of TRAIP function leads to enhanced chromosomal instability and decreased cell survival after replication stress. These findings establish TRAIP as a PCNA-binding ubiquitin ligase with an important role in protecting genome integrity after obstacles to DNA replication.     

Mrc1 is required for normal progression of replication forks throughout chromatin in S. cerevisiae

Mrc1 associates with replication forks, where it transmits replication stress signals and is required for normal replisome pausing in response to nucleotide depletion. Mrc1 also plays a poorly understood role in DNA replication, which appears distinct from its role in checkpoint signaling. Here, we demonstrate that Mrc1 functions constitutively to promote normal replication fork progression. In mrc1Delta cells, replication forks proceed slowly throughout chromatin, rather than being specifically defective in pausing and progression through loci that impede fork progression. Analysis of genetic interactions with Rrm3, a DNA helicase required to resolve paused forks, indicates that Mrc1 checkpoint signaling is dispensable for the resolution of stalled replication forks and suggests that replication forks lacking Mrc1 create DNA damage that must be repaired by Rrm3. These findings elucidate a central role for Mrc1 in normal replisome function, which is distinct from its role as a checkpoint mediator, but nevertheless critical to genome stability.     

Checkpoint-dependent and -independent roles of Swi3 in replication fork recovery and sister chromatid cohesion in fission yeast

Multiple genome maintenance processes are coordinated at the replication fork to preserve genomic integrity. How eukaryotic cells accomplish such a coordination is unknown. Swi1 and Swi3 form the replication fork protection complex and are involved in various processes including stabilization of replication forks, activation of the Cds1 checkpoint kinase and establishment of sister chromatid cohesion in fission yeast. However, the mechanisms by which the Swi1-Swi3 complex achieves and coordinates these tasks are not well understood. Here, we describe the identification of separation-of-function mutants of Swi3, aimed at dissecting the molecular pathways that require Swi1-Swi3. Unlike swi3 deletion mutants, the separation-of-function mutants were not sensitive to agents that stall replication forks. However, they were highly sensitive to camptothecin that induces replication fork breakage. In addition, these mutants were defective in replication fork regeneration and sister chromatid cohesion. Interestingly, unlike swi3-deleted cell, the separation-of-functions mutants were proficient in the activation of the replication checkpoint, but their fork regeneration defects were more severe than those of checkpoint mutants including cds1Δ, chk1Δ and rad3Δ. These results suggest that, while Swi3 mediates full activation of the replication checkpoint in response to stalled replication forks, Swi3 activates a checkpoint-independent pathway to facilitate recovery of collapsed replication forks and the establishment of sister chromatid cohesion. Thus, our separation-of-function alleles provide new insight into understanding the multiple roles of Swi1-Swi3 in fork protection during DNA replication, and into understanding how replication forks are maintained in response to different genotoxic agents.     

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.     

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.