The checkpoint response to replication stress

Genome instability is a hallmark of cancer cells, and defective DNA replication, repair and recombination have been linked to its etiology. Increasing evidence suggests that proteins influencing S-phase processes such as replication fork movement and stability, repair events and replication completion, have significant roles in maintaining genome stability. DNA damage and replication stress activate a signal transduction cascade, often referred to as the checkpoint response. A central goal of the replication checkpoint is to maintain the integrity of the replication forks while facilitating replication completion and DNA repair and coordinating these events with cell cycle transitions. Progression through the cell cycle in spite of defective or incomplete DNA synthesis or unrepaired DNA lesions may result in broken chromosomes, genome aberrations, and an accumulation of mutations. In this review we discuss the multiple roles of the replication checkpoint during replication and in response to replication stress, as well as the enzymatic activities that cooperate with the checkpoint pathway to promote fork resumption and repair of DNA lesions thereby contributing to genome integrity.     

DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation,fork restart,homologous recombination and mitotic catastrophe

Genotoxins and other factors cause replication stress that activate the DNA damage response (DDR), comprising checkpoint and repair systems. The DDR suppresses cancer by promoting genome stability, and it regulates tumor resistance to chemo- and radiotherapy. Three members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM, ATR, and DNA-PK, are important DDR proteins. A key PIKK target is replication protein A (RPA), which binds single-stranded DNA and functions in DNA replication, DNA repair, and checkpoint signaling. An early response to replication stress is ATR activation, which occurs when RPA accumulates on ssDNA. Activated ATR phosphorylates many targets, including the RPA32 subunit of RPA, leading to Chk1 activation and replication arrest. DNA-PK also phosphorylates RPA32 in response to replication stress, and we demonstrate that cells with DNA-PK defects, or lacking RPA32 Ser4/Ser8 targeted by DNA-PK, confer similar phenotypes, including defective replication checkpoint arrest, hyper-recombination, premature replication fork restart, failure to block late origin firing, and increased mitotic catastrophe. We present evidence that hyper-recombination in these mutants is ATM-dependent, but the other defects are ATM-independent. These results indicate that DNA-PK and ATR signaling through RPA32 plays a critical role in promoting genome stability and cell survival in response to replication stress.     

Separable functions of Tof1/Timeless in intra-S-checkpoint signalling,replisome stability and DNA topological stress

The highly conserved Tof1/Timeless proteins minimise replication stress and promote normal DNA replication. They are required to mediate the DNA replication checkpoint (DRC), the stable pausing of forks at protein fork blocks, the coupling of DNA helicase and polymerase functions during replication stress (RS) and the preferential resolution of DNA topological stress ahead of the fork. Here we demonstrate that the roles of the Saccharomyces cerevisiae Timeless protein Tof1 in DRC signalling and resolution of DNA topological stress require distinct N and C terminal regions of the protein, whereas the other functions of Tof1 are closely linked to the stable interaction between Tof1 and its constitutive binding partner Csm3/Tipin. By separating the role of Tof1 in DRC from fork stabilisation and coupling, we show that Tof1 has distinct activities in checkpoint activation and replisome stability to ensure the viable completion of DNA replication following replication stress.     

Pol32 is required for Pol zeta-dependent translesion synthesis and prevents double-strand breaks at the replication fork

POL32 encodes a non-essential subunit of Polδ and plays a role in Polδ processivity and DNA repair. In order to understand how Pol32 is involved in these processes, we performed extensive genetic analysis and demonstrated that POL32 is required for Polζ-mediated translesion synthesis, but not for Polη-mediated activity. Unlike Polζ, inactivation of Pol32 does not result in decreased spontaneous mutagenesis, nor does it limit genome instability in the absence of the error-free postreplication repair pathway. In contrast, inactivation of Pol32 results in an increased rate of replication slippage and recombination. A genome-wide synthetic lethal screen revealed that in the absence of Pol32, homologous recombination repair and cell cycle checkpoints play crucial roles in maintaining cell survival and growth. These results are consistent with a model in which Pol32 functions as a coupling factor to facilitate a switch from replication to translesion synthesis when Polδ encounters replication-blocking lesions. When Pol32 is absent, the S-phase checkpoint complex Mrc1–Tof1 becomes crucial to stabilize the stalled replication fork and recruit Top3 and Sgs1. Lack of any of the above activities will cause double strand breaks at or near the replication fork that require recombination as well as Rad9 for cell survival.     

The F-box protein Dia2 overcomes replication impedance to promote genome stability in Saccharomyces cerevisiae

The maintenance of DNA replication fork stability under conditions of DNA damage and at natural replication pause sites is essential for genome stability. Here, we describe a novel role for the F-box protein Dia2 in promoting genome stability in the budding yeast Saccharomyces cerevisiae. Like most other F-box proteins, Dia2 forms a Skp1-Cdc53/Cullin-F-box (SCF) E3 ubiquitin–ligase complex. Systematic analysis of genetic interactions between dia2Δ and ~4400 viable gene deletion mutants revealed synthetic lethal/synthetic sick interactions with a broad spectrum of DNA replication, recombination, checkpoint, and chromatin-remodeling pathways. dia2Δ strains exhibit constitutive activation of the checkpoint kinase Rad53 and elevated counts of endogenous DNA repair foci and are unable to overcome MMS-induced replicative stress. Notably, dia2Δ strains display a high rate of gross chromosomal rearrangements (GCRs) that involve the rDNA locus and an increase in extrachromosomal rDNA circle (ERC) formation, consistent with an observed enrichment of Dia2 in the nucleolus. These results suggest that Dia2 is essential for stable passage of replication forks through regions of damaged DNA and natural fragile regions, particularly the replication fork barrier (RFB) of rDNA repeat loci. We propose that the SCF^Dia2 ubiquitin ligase serves to modify or degrade protein substrates that would otherwise impede the replication fork in problematic regions of the genome.     

A Dbf4 mutant contributes to bypassing the Rad53-mediated block of origins of replication in response to genotoxic stress

An intra-S phase checkpoint slows the rate of DNA replication in response to DNA damage and replication fork blocks in eukaryotic cells. In the budding yeast Saccharomyces cerevisiae, such down-regulation is achieved through the Rad53 kinase-dependent block of origins of replication. We have identified the Rad53 phosphorylation sites on Dbf4, the activator subunit of the essential S phase Dbf4-dependent kinase, and generated a non-phosphorylatable Dbf4 mutant (dbf4(7A)). We show here that dbf4(7A) is a bona fide intra-S phase checkpoint bypass allele that contributes to abrogating the Rad53 block of origin firing in response to genotoxic stress.     

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.     

The amino terminus of the Saccharomyces cerevisiae DNA helicase Rrm3p modulates protein function altering replication and checkpoint activity

The Pif1 family of DNA helicases is conserved from yeast to humans. Although the helicase domains of family members are well conserved, the amino termini of these proteins are not. The Saccharomyces cerevisiae genome encodes two Pif1 family members, Rrm3p and Pif1p, that have very different functions. To determine if the amino terminus of Rrm3p contributes to its role in promoting fork progression at >1000 discrete chromosomal sites, we constructed a deletion series that lacked portions of the 249-amino-acid amino terminus. The phenotypes of cells expressing alleles that lacked all or most of the amino terminus were indistinguishable from those of rrm3Delta cells. Rrm3p deletion derivatives that lacked smaller portions of the amino terminus were also defective, but the extent of replication pausing at tRNA genes, telomeres, and ribosomal DNA (rDNA) was not as great as in rrm3Delta cells. Deleting only 62 amino acids from the middle of the amino terminus affected only rDNA replication, suggesting that the amino terminus can confer locus-specific effects. Cells expressing a fusion protein consisting of the Rrm3p amino terminus and the Pif1p helicase domain displayed defects similar to rrm3Delta cells. These data demonstrate that the amino terminus of Rrm3p is essential for Rrm3p function. However, the helicase domain of Rrm3p also contributes to its functional specificity.     

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.     

Schizosaccharomyces pombe minichromosome maintenance-binding protein (MCM-BP) antagonizes MCM helicase

The minichromosome maintenance (MCM) complex, a replicative helicase, is a heterohexamer essential for DNA duplication and genome stability. We identified Schizosaccharomyces pombe mcb1(+) (Mcm-binding protein 1), an apparent orthologue of the human MCM-binding protein that associates with a subset of MCM complex proteins. mcb1(+) is an essential gene. Deletion of mcb1(+) caused cell cycle arrest after several generations with a cdc phenotype and disrupted nuclear structure. Mcb1 is an abundant protein, constitutively present across the cell cycle. It is widely distributed in cytoplasm and nucleoplasm and bound to chromatin. Co-immunoprecipitation suggested that Mcb1 interacts robustly with Mcm3-7 but not Mcm2. Overproduction of Mcb1 disrupted the association of Mcm2 with other MCM proteins, resulting in inhibition of DNA replication, DNA damage, and activation of the checkpoint kinase Chk1. Thus, Mcb1 appears to antagonize the function of MCM helicase.     

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.     

Replication Fork Stalling and Checkpoint Activation by a PKD1 Locus Mirror Repeat Polypurine-Polypyrimidine (Pu-Py) Tract

DNA sequences prone to forming noncanonical structures (hairpins, triplexes, G-quadruplexes) cause DNA replication fork stalling, activate DNA damage responses, and represent hotspots of genomic instability associated with human disease. The 88-bp asymmetric polypurine-polypyrimidine (Pu-Py) mirror repeat tract from the human polycystic kidney disease (PKD1) intron 21 forms non-B DNA secondary structures in vitro. We show that the PKD1 mirror repeat also causes orientation-dependent fork stalling during replication in vitro and in vivo. When integrated alongside the c-myc replicator at an ectopic chromosomal site in the HeLa genome, the Pu-Py mirror repeat tract elicits a polar replication fork barrier. Increased replication protein A (RPA), Rad9, and ataxia telangiectasia- and Rad3-related (ATR) checkpoint protein binding near the mirror repeat sequence suggests that the DNA damage response is activated upon replication fork stalling. Moreover, the proximal c-myc origin of replication was not required to cause orientation-dependent checkpoint activation. Cells expressing the replication fork barrier display constitutive Chk1 phosphorylation and continued growth, i.e. checkpoint adaptation. Excision of the Pu-Py mirror repeat tract abrogates the DNA damage response. Adaptation to Chk1 phosphorylation in cells expressing the replication fork barrier may allow the accumulation of mutations that would otherwise be remediated by the DNA damage response.     

A whole genome RNAi screen identifies replication stress response genes

Proper DNA replication is critical to maintain genome stability. When the DNA replication machinery encounters obstacles to replication, replication forks stall and the replication stress response is activated. This response includes activation of cell cycle checkpoints, stabilization of the replication fork, and DNA damage repair and tolerance mechanisms. Defects in the replication stress response can result in alterations to the DNA sequence causing changes in protein function and expression, ultimately leading to disease states such as cancer. To identify additional genes that control the replication stress response, we performed a three-parameter, high content, whole genome siRNA screen measuring DNA replication before and after a challenge with replication stress as well as a marker of checkpoint kinase signalling. We identified over 200 replication stress response genes and subsequently analyzed how they influence cellular viability in response to replication stress. These data will serve as a useful resource for understanding the replication stress response.     

Saccharomyces cerevisiae Rrm3p DNA helicase promotes genome integrity by preventing replication fork stalling: viability of rrm3 cells requires the intra-S-phase checkpoint and fork restart activities

Rrm3p is a 5'-to-3' DNA helicase that helps replication forks traverse protein-DNA complexes. Its absence leads to increased fork stalling and breakage at over 1,000 specific sites located throughout the Saccharomyces cerevisiae genome. To understand the mechanisms that respond to and repair rrm3-dependent lesions, we carried out a candidate gene deletion analysis to identify genes whose mutation conferred slow growth or lethality on rrm3 cells. Based on synthetic phenotypes, the intra-S-phase checkpoint, the SRS2 inhibitor of recombination, the SGS1/TOP3 replication fork restart pathway, and the MRE11/RAD50/XRS2 (MRX) complex were critical for viability of rrm3 cells. DNA damage checkpoint and homologous recombination genes were important for normal growth of rrm3 cells. However, the MUS81/MMS4 replication fork restart pathway did not affect growth of rrm3 cells. These data suggest a model in which the stalled and broken forks generated in rrm3 cells activate a checkpoint response that provides time for fork repair and restart. Stalled forks are converted by a Rad51p-mediated process to intermediates that are resolved by Sgs1p/Top3p. The rrm3 system provides a unique opportunity to learn the fate of forks whose progress is impaired by natural impediments rather than by exogenous DNA damage.     

Control of translocations between highly diverged genes by Sgs1, the Saccharomyces cerevisiae homolog of the Bloom's syndrome protein

Sgs1 is a RecQ family DNA helicase required for genome stability in Saccharomyces cerevisiae whose human homologs BLM, WRN, and RECQL4 are mutated in Bloom's, Werner, and Rothmund Thomson syndromes, respectively. Sgs1 and mismatch repair (MMR) are inhibitors of recombination between similar but divergent (homeologous) DNA sequences. Here we show that SGS1, but not MMR, is critical for suppressing spontaneous, recurring translocations between diverged genes in cells with mutations in the genes encoding the checkpoint proteins Mec3, Rad24, Rad9, or Rfc5, the chromatin assembly factors Cac1 or Asf1, and the DNA helicase Rrm3. The S-phase checkpoint kinase and telomere maintenance factor Tel1, a homolog of the human ataxia telangiectasia (ATM) protein, prevents these translocations, whereas the checkpoint kinase Mec1, a homolog of the human ATM-related protein, and the Rad53 checkpoint kinase are not required. The translocation structures observed suggest involvement of a dicentric intermediate and break-induced replication with multiple cycles of DNA template switching.     

Mrc1 and Tof1 promote replication fork progression and recovery independently of Rad53

The yeast checkpoint factors Mrc1p and Tof1p travel with the replication fork and mediate the activation of the Rad53p kinase in response to a replication stress. We show here that both proteins are required for normal fork progression but play different roles at stalled forks. Tof1p is critical for the activity of the rDNA replication fork barrier (RFB) but plays a minor role in the replication checkpoint. In contrast, Mrc1p is not necessary for RFB activity but is essential to mediate the replication stress response. Interestingly, stalled forks did not collapse in mrc1Delta cells exposed to hydroxyurea (HU) as they do in rad53 mutants. However, forks failed to restart when mrc1Delta cells were released from the block. The critical role of Mrc1p in HU is therefore to promote fork recovery in a Rad53p-independent manner, presumably through the formation of a stable fork-pausing complex.     

Hsk1-Dfp1/Him1, the Cdc7-Dbf4 kinase in Schizosaccharomyces pombe, associates with Swi1, a component of the replication fork protection complex

The protein kinase Hsk1 is essential for DNA replication in Schizosaccharomyces pombe. It associates with Dfp1/Him1 to form an active complex equivalent to the Cdc7-Dbf4 protein kinase in Saccharomyces cerevisiae. Swi1 and Swi3 are subunits of the replication fork protection complex in S. pombe that is homologous to the Tof1-Csm3 complex in S. cerevisiae. The fork protection complex helps to preserve the integrity of stalled replication forks and is important for activation of the checkpoint protein kinase Cds1 in response to fork arrest. Here we describe physical and genetic interactions involving Swi1 and Hsk1-Dfp1/Him1. Dfp1/Him1 was identified in a yeast two-hybrid screen with Swi1. Hsk1 and Dfp1/Him1 both co-immunoprecipitate with Swi1. Swi1 is required for growth of a temperature-sensitive hsk1 (hsk1ts) mutant at its semi-permissive temperature. Hsk1ts cells accumulate Rad22 (Rad52 homologue) DNA repair foci at the permissive temperature, as previously observed in swi1 cells, indicating that abnormal single-stranded DNA regions form near the replication fork in hsk1ts cells. hsk1ts cells were also unable to properly delay S-phase progression in the presence of a DNA alkylating agent and were partially defective in mating type switching. These data suggest that Hsk1-Dfp1/Him1 and Swi1-Swi3 complexes have interrelated roles in stabilization of arrested replication forks.     

Csm3, Tof1, and Mrc1 Form a Heterotrimeric Mediator Complex That Associates with DNA Replication Forks

Mrc1 (mediator of replication checkpoint), Tof1 (topoisomerase I interacting factor), and Csm3 (chromosome segregation in meiosis) are checkpoint-mediator proteins that function during DNA replication and activate the effector kinase Rad53. We reported previously that Mrc1 and Tof1 are constituents of the replication machinery and that both proteins are required for the proper arrest and stabilization of replication forks in the presence of hydroxyurea. In our current study, we show that Csm3 is a component of moving replication forks and that both Tof1 and Csm3 are specifically required for the association of Mrc1 with these structures. In contrast, the deletion of mrc1 did not affect the association of Tof1 and Csm3 with the replication fork complex. In agreement with previous observations in yeast cells, the results of a baculovirus coexpression system showed that these three proteins interact directly with each other to form a mediator complex in the absence of replication forks.     

Fission yeast Swi1-Swi3 complex facilitates DNA binding of Mrc1

Replication fork protection complex Swi1-Swi3 and replication checkpoint mediator Mrc1 are required for maintenance of replication fork integrity during the course of DNA replication in the fission yeast Schizosaccharomyces pombe. These proteins play crucial roles in stabilizing stalled forks and activating replication checkpoint signaling pathways. Although they are conserved replication fork components, precise biochemical roles of these proteins are not known. Here we purified Mrc1 and Swi1-Swi3 proteins and show that these proteins bind to DNA independently but synergistically in vitro. Mrc1 binds preferentially to arrested fork or D-loop-like structures, although the affinity is relatively low, whereas the Swi1-Swi3 complex binds to double-stranded DNA with higher affinity. In the presence of a low concentration of Swi1-Swi3, Mrc1 generates a novel ternary complex and binds to various types of DNA with higher affinity. Moreover, purified Mrc1 and Swi1-Swi3 physically interact with each other, and this interaction is lost by mutations in the known DNA binding domain of Mrc1 (K235E,K236E). The interaction is also lost in a mutant form of Swi1 (E662K) that is specifically defective in polar fork arrest at a site called RTS1 and causes sensitivity to genotoxic agents, although the DNA binding affinity of Swi1-Swi3 is not affected by this mutation. As expected, the synergistic effect of the Swi1-Swi3 on DNA binding of Mrc1 is also lost by these mutations affecting the interaction between Mrc1 and Swi1-Swi3. Our results reveal an aspect of molecular interactions that may play an important role in replication pausing and fork stabilization.