Local and global functions of Timeless and Tipin in replication fork protection

The eukaryotic cell replicates its chromosomal DNA with almost absolute fidelity in the course of every cell cycle. This accomplishment is remarkable considering that the conditions for DNA replication are rarely ideal. The replication machinery encounters a variety of obstacles on the chromosome, including damaged template DNA. In addition, a number of chromosome regions are considered to be difficult to replicate owing to DNA secondary structures and DNA binding proteins required for various transactions on the chromosome. Under these conditions, replication forks stall or break, posing grave threats to genomic integrity. How does the cell combat such stressful conditions during DNA replication? The replication fork protection complex (FPC) may help answer this question. Recent studies have demonstrated that the FPC is required for the smooth passage of replication forks at difficult-to-replicate genomic regions and plays a critical role in coordinating multiple genome maintenance processes at the replication fork.     

Sap1 promotes the association of the replication fork protection complex with chromatin and is involved in the replication checkpoint in Schizosaccharomyces pombe

Sap1 is involved in replication fork pausing at rDNA repeats and functions during mating-type switching in Schizosaccharomyces pombe. These two roles are dependent on the ability of Sap1 to bind specific DNA sequences at the rDNA and mating-type loci, respectively. In S. pombe, Swi1 and Swi3 form the replication fork protection complex (FPC) and play important roles in the activation of the replication checkpoint and the stabilization of stalled replication forks. Here we describe the roles of Sap1 in the replication checkpoint. We show that Sap1 is involved in the activation of the replication checkpoint kinase Cds1 and that sap1 mutant cells accumulate spontaneous DNA damage during the S- and G2-phases, which is indicative of fork damage. We also show that sap1 mutants have a defect in the resumption of DNA replication after fork arrest. Sap1 is localized at the replication origin ori2004 and this localization is required for the association of the FPC with chromatin. We propose that Sap1 is required to recruit the FPC to chromatin, thereby contributing to the activation of the replication checkpoint and the stabilization of 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．     

Orchestration of the S-phase and DNA damage checkpoint pathways by replication forks from early origins

The S-phase checkpoint activated at replication forks coordinates DNA replication when forks stall because of DNA damage or low deoxyribonucleotide triphosphate pools. We explore the involvement of replication forks in coordinating the S-phase checkpoint using dun1Delta cells that have a defect in the number of stalled forks formed from early origins and are dependent on the DNA damage Chk1p pathway for survival when replication is stalled. We show that providing additional origins activated in early S phase and establishing a paused fork at a replication fork pause site restores S-phase checkpoint signaling to chk1Delta dun1Delta cells and relieves the reliance on the DNA damage checkpoint pathway. Origin licensing and activation are controlled by the cyclin-Cdk complexes. Thus, oncogene-mediated deregulation of cyclins in the early stages of cancer development could contribute to genomic instability through a deficiency in the forks required to establish the S-phase checkpoint.     

The MRX complex stabilizes the replisome independently of the S phase checkpoint during replication stress

The Mre11–Rad50–Xrs2 (MRX) complex has an important function in the maintenance of genomic integrity by contributing to the detection and repair of chromosome breaks. Here we show that the complex is recruited to sites of paused forks where it stabilizes the association of essential replisome components. Interestingly, this function is not dependent on the S phase checkpoint or the nuclease activity of Mre11. We find that disruption of the MRX complex leads to a loss of fork recovery and a failure to properly complete DNA replication when cells are exposed to replication stress. Our data suggest that one critical function of the MRX complex during replication is to promote the cohesion of sister chromatids at paused forks, offering an explanation for why MRX deficiency leads to a loss of cell viability and high levels of chromosome rearrangements under conditions of replication stress.     

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.     

The cullin Rtt101p promotes replication fork progression through damaged DNA and natural pause sites

Accurate and complete DNA replication is fundamental to maintain genome integrity. While the mechanisms and underlying machinery required to duplicate bulk genomic DNA are beginning to emerge, little is known about how cells replicate through damaged areas and special chromosomal regions such as telomeres, centromeres, and highly transcribed loci . Here, we have investigated the role of the yeast cullin Rtt101p in this process. We show that rtt101Delta cells accumulate spontaneous DNA damage and exhibit a G(2)/M delay, even though they are fully proficient to detect and repair chromosome breaks. Viability of rtt101Delta mutants depends on Rrm3p, a DNA helicase involved in displacing proteinaceous complexes at programmed pause sites . Moreover, rtt101Delta cells show hyperrecombination at forks arrested at replication fork barriers (RFBs) of ribosomal DNA. Finally, rtt101Delta mutants are sensitive to fork arrest induced by DNA alkylation, but not by nucleotide depletion. We therefore propose that the cullin Rtt101p promotes fork progression through obstacles such as DNA lesions or tightly bound protein-DNA complexes via a new mechanism involving ubiquitin-conjugation.     

Pathological replication in cells lacking RecG DNA translocase

Little is known about what happens when forks meet to complete DNA replication in any organism. In this study we present data suggesting that the collision of replication forks is a potential threat to genomic stability. We demonstrate that Escherichia coli cells lacking RecG helicase suffer major defects in chromosome replication following UV irradiation, and that this is associated with high levels of DNA synthesis initiated independently of the initiator protein DnaA. This UV-induced stable DNA replication is dependent on PriA helicase and continues long after UV-induced lesions have been excised. We suggest UV irradiation triggers the assembly of new replication forks, leading to multiple fork collisions outside the terminus area. Such collisions may generate branched DNAs that serve to establish further new forks, resulting in uncontrolled DNA amplification. We propose that RecG reduces the likelihood of this pathological cascade being set in motion by reducing initiation of replication at D- and R-loops, and other structures generated as a result of fork collisions. Our results shed light on why replication initiation in bacteria is limited to a single origin and why termination is carefully orchestrated to a single event within a restricted area each cell cycle.     

Multiple pathways cooperate to facilitate DNA replication fork progression through alkylated DNA

Eukaryotic genomes are especially vulnerable to DNA damage during the S phase of the cell cycle, when chromosomes must be duplicated. The stability of DNA replication forks is critical to achieve faithful chromosome replication and is severely compromised when forks encounter DNA lesions. To maintain genome integrity, replication forks need to be protected by the S-phase checkpoint and DNA insults must be repaired. Different pathways help to repair or tolerate the lesions in the DNA, but their contribution to the progression of replication forks through damaged DNA is not well known. Here we show in budding yeast that, when the DNA template is damaged with the alkylating agent methyl methanesulfonate (MMS), base excision repair, homologous recombination and DNA damage tolerance pathways, together with a functional S-phase checkpoint, are essential for the efficient progression of DNA replication forks and the maintenance of cell survival. In the absence of base excision repair, replication forks stall reversibly in cells exposed to MMS. This repair reaction is necessary to eliminate the lesions that impede fork progression and has to be coordinated with recombination and damage tolerance activities to avoid fork collapse and allow forks to resume and complete chromosome replication.     

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.     

Dysregulation of DNA polymerase κ recruitment to replication forks results in genomic instability

Translesion synthesis polymerases (TLS Pols) are required to tolerate DNA lesions that would otherwise cause replication arrest and cell death. Aberrant expression of these specialized Pols may be responsible for increased mutagenesis and loss of genome integrity in human cancers. The molecular events that control the usage of TLS Pols in non-pathological conditions remain largely unknown. Here, we show that aberrant recruitment of TLS Polκ to replication forks results in genomic instability and can be mediated through the loss of the deubiquitinase USP1. Moreover, artificial tethering of Polκ to proliferating cell nuclear antigen (PCNA) circumvents the need for its ubiquitin-binding domain in the promotion of genomic instability. Finally, we show that the loss of USP1 leads to a dramatic reduction of replication fork speed in a Polκ-dependent manner. We propose a mechanism whereby reversible ubiquitination of PCNA can prevent spurious TLS Pol recruitment and regulate replication fork speed to ensure the maintenance of genome integrity.     

PARP is activated at stalled forks to mediate Mre11‐dependent replication restart and recombination

If replication forks are perturbed, a multifaceted response including several DNA repair and cell cycle checkpoint pathways is activated to ensure faithful DNA replication. Here, we show that poly(ADP‐ribose) polymerase 1 (PARP1) binds to and is activated by stalled replication forks that contain small gaps. PARP1 collaborates with Mre11 to promote replication fork restart after release from replication blocks, most likely by recruiting Mre11 to the replication fork to promote resection of DNA. Both PARP1 and PARP2 are required for hydroxyurea‐induced homologous recombination to promote cell survival after replication blocks. Together, our data suggest that PARP1 and PARP2 detect disrupted replication forks and attract Mre11 for end processing that is required for subsequent recombination repair and restart of replication forks.     

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.     

Replication fork stalling by bulky DNA damage: localization at active origins and checkpoint modulation

The integrity of the genome is threatened by DNA damage that blocks the progression of replication forks. Little is known about the genomic locations of replication fork stalling, and its determinants and consequences in vivo. Here we show that bulky DNA damaging agents induce localized fork stalling at yeast replication origins, and that localized stalling is dependent on proximal origin activity and is modulated by the intra-S-phase checkpoint. Fork stalling preceded the formation of sister chromatid junctions required for bypassing DNA damage. Despite DNA adduct formation, localized fork stalling was abrogated at an origin inactivated by a point mutation and prominent stalling was not detected at naturally-inactive origins in the replicon. The intra-S-phase checkpoint contributed to the high-level of fork stalling at early origins, while checkpoint inactivation led to initiation, localized stalling and chromatid joining at a late origin. Our results indicate that replication forks initially encountering a bulky DNA adduct exhibit a dual nature of stalling: a checkpoint-independent arrest that triggers sister chromatid junction formation, as well as a checkpoint-enhanced arrest at early origins that accompanies the repression of late origin firing. We propose that the initial checkpoint-enhanced arrest reflects events that facilitate fork resolution at subsequent lesions.     

Regulation of replication fork speed: Mechanisms and impact on genomic stability

Replication of DNA is a fundamental biological process that ensures precise duplication of the genome and thus safeguards inheritance. Any errors occurring during this process must be repaired before the cell divides, by activating the DNA damage response (DDR) machinery that detects and corrects the DNA lesions. Consistent with its significance, DNA replication is under stringent control, both spatial and temporal. Defined regions of the genome are replicated at specific times during S phase and the speed of replication fork progression is adjusted to fully replicate DNA in pace with the cell cycle. Insults that impair DNA replication cause replication stress (RS), which can lead to genomic instability and, potentially, to cell transformation. In this perspective, we review the current concept of replication stress, including the recent findings on the effects of accelerated fork speed and their impact on genomic (in)stability. We discuss in detail the Fork Speed Regulatory Network (FSRN), an integrated molecular machinery that regulates the velocity of DNA replication forks. Finally, we explore the potential for targeting FSRN components as an avenue to treat cancer.     

Replication fork dynamics and the DNA damage response

Prevention and repair of DNA damage is essential for maintenance of genomic stability and cell survival. DNA replication during S-phase can be a source of DNA damage if endogenous or exogenous stresses impair the progression of replication forks. It has become increasingly clear that DNA-damage-response pathways do not only respond to the presence of damaged DNA, but also modulate DNA replication dynamics to prevent DNA damage formation during S-phase. Such observations may help explain the developmental defects or cancer predisposition caused by mutations in DNA-damage-response genes. The present review focuses on molecular mechanisms by which DNA-damage-response pathways control and promote replication dynamics in vertebrate cells. In particular, DNA damage pathways contribute to proper replication by regulating replication initiation, stabilizing transiently stalled forks, promoting replication restart and facilitating fork movement on difficult-to-replicate templates. If replication fork progression fails to be rescued, this may lead to DNA damage and genomic instability via nuclease processing of aberrant fork structures or incomplete sister chromatid separation during mitosis.     

Proteasome-dependent degradation of replisome components regulates faithful DNA replication

The replication machinery, or the replisome, collides with a variety of obstacles during the normal process of DNA replication. In addition to damaged template DNA, numerous chromosome regions are considered to be difficult to replicate owing to the presence of DNA secondary structures and DNA-binding proteins. Under these conditions, the replication fork stalls, generating replication stress. Stalled forks are prone to collapse, posing serious threats to genomic integrity. It is generally thought that the replication checkpoint functions to stabilize the replisome and replication fork structure upon replication stress. This is important in order to allow DNA replication to resume once the problem is solved. However, our recent studies demonstrated that some replisome components undergo proteasome-dependent degradation during DNA replication in the fission yeast Schizosaccharomyces pombe. Our investigation has revealed the involvement of the SCF^Pof3 (Skp1-Cullin/Cdc53-F-box) ubiquitin ligase in replisome regulation. We also demonstrated that forced accumulation of the replisome components leads to abnormal DNA replication upon replication stress. Here we review these findings and present additional data indicating the importance of replisome degradation for DNA replication. Our studies suggest that cells activate an alternative pathway to degrade replisome components in order to preserve genomic integrity.     

Recovery of Arrested Replication Forks by Homologous Recombination Is Error-Prone

Homologous recombination is a universal mechanism that allows repair of DNA and provides support for DNA replication. Homologous recombination is therefore a major pathway that suppresses non-homology-mediated genome instability. Here, we report that recovery of impeded replication forks by homologous recombination is error-prone. Using a fork-arrest-based assay in fission yeast, we demonstrate that a single collapsed fork can cause mutations and large-scale genomic changes, including deletions and translocations. Fork-arrest-induced gross chromosomal rearrangements are mediated by inappropriate ectopic recombination events at the site of collapsed forks. Inverted repeats near the site of fork collapse stimulate large-scale genomic changes up to 1,500 times over spontaneous events. We also show that the high accuracy of DNA replication during S-phase is impaired by impediments to fork progression, since fork-arrest-induced mutation is due to erroneous DNA synthesis during recovery of replication forks. The mutations caused are small insertions/duplications between short tandem repeats (micro-homology) indicative of replication slippage. Our data establish that collapsed forks, but not stalled forks, recovered by homologous recombination are prone to replication slippage. The inaccuracy of DNA synthesis does not rely on PCNA ubiquitination or trans-lesion-synthesis DNA polymerases, and it is not counteracted by mismatch repair. We propose that deletions/insertions, mediated by micro-homology, leading to copy number variations during replication stress may arise by progression of error-prone replication forks restarted by homologous recombination.     

真核细胞DNA复制叉稳定性维持机制的研究进展

DNA复制是细胞最基本的生命活动之一,是生物体生存和繁殖的基础。从原核生物到真核生物,DNA复制过程基本保守,分为复制起始和延伸两个阶段。复制叉是DNA复制的基本结构,它容易遭受多种内源或外源的DNA复制压力影响而停顿,导致基因组不稳定,引起细胞凋亡、癌变或细胞死亡等严重后果。为了维持复制叉的稳定,细胞进化出了一系列机制,其中最重要机制之一便是S期细胞周期检验点。就影响DNA复制叉稳定的内外因素、S期细胞周期检验点与复制叉稳定性的关系以及复制叉稳定性与相关疾病的发生、治疗等问题进行简要综述。