In vivo labeling of fission yeast DNA with thymidine and thymidine analogs

In vivo labeling of DNA with thymidine and thymidine analogs has long been a cornerstone of replication studies. Unfortunately, yeast lack a thymidine salvage pathway and thus do not incorporate exogenous thymidine. Specifically, yeast neither efficiently take up exogenous thymidine from their growth media nor phosphorylate it to thymidylate, the precursor of dTTP. We have overcome these problems in fission yeast by expressing the human equilibrative nucleoside transporter 1 (hENT1) along with herpes simplex virus thymidine kinase (tk). hENT1 tk cells are healthy and efficiently incorporate exogenous thymidine and thymidine analogs. We present protocols for labeling DNA with tritiated thymidine, for in situ detection of incorporated BrdU by immunofluorescence, for double labeling with CldU and IdU, for CsCl gradient separation of IdU-labeled DNA, and for using hENT1 and tk as both positive and negative selection markers.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is unable to incorporate exogenous nucleosides into DNA. We have made a number of improvements to existing strategies to reconstitute an efficient thymidine salvage pathway in yeast. We have constructed strains that express both a nucleoside kinase as well as an equilibrative nucleoside transporter. By also deleting the gene encoding thymidylate synthase (CDC21) we have constructed strains that are entirely dependent upon exogenous thymidine for viability and that can grow with normal kinetics at low thymidine concentrations. Using this novel approach, we show that depletion of a single deoxyribonucleoside causes reversible arrest of cells in S phase with concomitant phosphorylation and activation of the S phase checkpoint kinase, Rad53. We show that this strain also efficiently incorporates the thymidine analogue, BrdU, into DNA and can be used for pulse–chase labelling.     

Protein kinase C controls activation of the DNA integrity checkpoint

The protein kinase C (PKC) superfamily plays key regulatory roles in numerous cellular processes. Saccharomyces cerevisiae contains a single PKC, Pkc1, whose main function is cell wall integrity maintenance. In this work, we connect the Pkc1 protein to the maintenance of genome integrity in response to genotoxic stresses. Pkc1 and its kinase activity are necessary for the phosphorylation of checkpoint kinase Rad53, histone H2A and Xrs2 protein after deoxyribonucleic acid (DNA) damage, indicating that Pkc1 is required for activation of checkpoint kinases Mec1 and Tel1. Furthermore, Pkc1 electrophoretic mobility is delayed after inducing DNA damage, which reflects that Pkc1 is post-translationally modified. This modification is a phosphorylation event mediated by Tel1. The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint. Finally, downregulation of PKCδ activity in HeLa cells caused a defective activation of checkpoint kinase Chk2 when DNA damage was induced. Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.     

Mus81, Rhp51(Rad51), and Rqh1 Form an Epistatic Pathway Required for the S-Phase DNA Damage Checkpoint

The S-phase DNA damage checkpoint slows the rate of DNA synthesis in response to damage during replication. In the fission yeast Schizosaccharomyces pombe, Cds1, the S-phase-specific checkpoint effector kinase, is required for checkpoint signaling and replication slowing; upon treatment with the alkylating agent methyl methane sulfonate, cds1Δ mutants display a complete checkpoint defect. We have identified proteins downstream of Cds1 required for checkpoint-dependant slowing, including the structure-specific endonuclease Mus81 and the helicase Rqh1, which are implicated in replication fork stability and the negative regulation of recombination. Removing Rhp51, the Rad51 recombinase homologue, suppresses the slowing defect of rqh1Δ mutants, but not that of mus81Δ mutant, defining an epistatic pathway in which mus81 is epistatic to rhp51 and rhp51 is epistatic to rqh1. We propose that restraining recombination is required for the slowing of replication in response to DNA damage.     

Behind the wheel and under the hood: Functions of cyclin-dependent kinases in response to DNA damage

Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair – homologous recombination (HR) and non-homologous end joining (NHEJ) – are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs – as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function – could be balanced to produce a coordinated and effective response to DNA damage.     

Cdc2 Tyrosine Phosphorylation is Not Required for the S-Phase DNA Damage Checkpoint in Fission Yeast

The S-phase DNA damage checkpoint slows replication when damage occurs during S phase. Cdc25, which activates Cdc2 by dephosphorylating tyrosine-15, has been shown to be a downstream target of the checkpoint in metazoans, but its role is not clear in fission yeast. The dephosphorylation of Cdc2 has been assumed not to play a role in S-phase regulation because cells replicate in the absence of Cdc25, demonstrating that tyrosine-15 phosphorylated Cdc2 is sufficient for S phase. However, it has been reported recently that Cdc25 is required for the slowing of S phase in response to damage in fission yeast, suggesting a modulatory role for Cdc2 dephosphorylation in S phase. We have investigated the role of Cdc25 and the tyrosine phosphorylation of Cdc2 in the S-phase damage checkpoint, and our results show that Cdc2 phosphorylation is not a target of the checkpoint. The checkpoint was not compromised in a Cdc25 overexpressing strain, a strain carrying non-phosphorylatable form of Cdc2, or in a strain lacking Cdc25. Our results are consistent with a strictly Cdc2-Y15 phosphorylation-independent mechanism of the fission yeast S-phase DNA damage checkpoint.     

Cdc2 phosphorylation of Crb2 is required for reestablishing cell cycle progression after the damage checkpoint.

DNA damage induces cell cycle arrest (called the damage checkpoint), during which cells carry out actions for repair. A fission yeast protein, Crb2/Rhp9, which resembles budding yeast Rad9p and human BRCA1, promotes checkpoint by activating Chk1 kinase, which restrains Cdc2 activation. We show here that phosphorylation of the T215 Cdc2 site of Crb2 is required for reentering the cell cycle after the damage-induced checkpoint arrest. If this site is nonphosphorylatable, irradiated cells remain arrested, though damage is repaired, and maintain the phosphorylated state of Chk1 kinase. The T215 site is in vitro phosphorylated by purified Cdc2 kinase. Phosphorylation of T215 occurs intensely in response to DNA damage at a late stage, suggesting an antagonistic role of Cdc2 phosphorylation toward checkpoint.     

Molecular Basis of the Essential S Phase Function of the Rad53 Checkpoint Kinase

The essential yeast kinases Mec1 and Rad53, or human ATR and Chk1, are crucial for checkpoint responses to exogenous genotoxic agents, but why they are also required for DNA replication in unperturbed cells remains poorly understood. Here we report that even in the absence of DNA-damaging agents, the rad53-4AQ mutant, lacking the N-terminal Mec1 phosphorylation site cluster, is synthetic lethal with a deletion of the RAD9 DNA damage checkpoint adaptor. This phenotype is caused by an inability of rad53-4AQ to activate the downstream kinase Dun1, which then leads to reduced basal deoxynucleoside triphosphate (dNTP) levels, spontaneous replication fork stalling, and constitutive activation of and dependence on S phase DNA damage checkpoints. Surprisingly, the kinase-deficient rad53-K227A mutant does not share these phenotypes but is rendered inviable by additional phosphosite mutations that prevent its binding to Dun1. The results demonstrate that ultralow Rad53 catalytic activity is sufficient for normal replication of undamaged chromosomes as long as it is targeted toward activation of the effector kinase Dun1. Our findings indicate that the essential S phase function of Rad53 is comprised by the combination of its role in regulating basal dNTP levels and its compensatory kinase function if dNTP levels are perturbed.     

The Role of MRN in the S-Phase DNA Damage Checkpoint Is Independent of Its Ctp1-dependent Roles in Double-Strand Break Repair and Checkpoint Signaling

The Mre11-Rad50-Nbs1 (MRN) complex has many biological functions: processing of double-strand breaks in meiosis, homologous recombination, telomere maintenance, S-phase checkpoint, and genome stability during replication. In the S-phase DNA damage checkpoint, MRN acts both in activation of checkpoint signaling and downstream of the checkpoint kinases to slow DNA replication. Mechanistically, MRN, along with its cofactor Ctp1, is involved in 5′ resection to create single-stranded DNA that is required for both signaling and homologous recombination. However, it is unclear whether resection is essential for all of the cellular functions of MRN. To dissect the various roles of MRN, we performed a structure–function analysis of nuclease dead alleles and potential separation-of-function alleles analogous to those found in the human disease ataxia telangiectasia-like disorder, which is caused by mutations in Mre11. We find that several alleles of rad32 (the fission yeast homologue of mre11), along with ctp1Δ, are defective in double-strand break repair and most other functions of the complex, but they maintain an intact S phase DNA damage checkpoint. Thus, the MRN S-phase checkpoint role is separate from its Ctp1- and resection-dependent role in double-strand break repair. This observation leads us to conclude that other functions of MRN, possibly its role in replication fork metabolism, are required for S-phase DNA damage checkpoint function.     

Meiotic S-phase damage activates recombination without checkpoint arrest

Checkpoints operate during meiosis to ensure the completion of DNA synthesis and programmed recombination before the initiation of meiotic divisions. Studies in the fission yeast Schizosaccharomyces pombe suggest that the meiotic response to DNA damage due to a failed replication checkpoint response differs substantially from the vegetative response, and may be influenced by the presence of homologous chromosomes. The checkpoint responses to DNA damage during fission yeast meiosis are not well characterized. Here we report that DNA damage induced during meiotic S-phase does not activate checkpoint arrest. We also find that in wild-type cells, markers for DNA breaks can persist at least to the first meiotic division. We also observe increased spontaneous S-phase damage in checkpoint mutants, which is repaired by recombination without activating checkpoint arrest. Our results suggest that fission yeast meiosis is exceptionally tolerant of DNA damage, and that some forms of spontaneous S-phase damage can be repaired by recombination without activating checkpoint arrest.     

Loss of Yeast Peroxiredoxin Tsa1p Induces Genome Instability through Activation of the DNA Damage Checkpoint and Elevation of dNTP Levels

Peroxiredoxins are a family of antioxidant enzymes critically involved in cellular defense and signaling. Particularly, yeast peroxiredoxin Tsa1p is thought to play a role in the maintenance of genome integrity, but the underlying mechanism is not understood. In this study, we took a genetic approach to investigate the cause of genome instability in tsa1Δ cells. Strong genetic interactions of TSA1 with DNA damage checkpoint components DUN1, SML1, and CRT1 were found when mutant cells were analyzed for either sensitivity to DNA damage or rate of spontaneous base substitutions. An elevation in intracellular dNTP production was observed in tsa1Δ cells. This was associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. In addition, defects in the DNA damage checkpoint did not modulate intracellular level of reactive oxygen species, but suppressed the mutator phenotype of tsa1Δ cells. On the contrary, overexpression of RNR1 exacerbated this phenotype by increasing dNTP levels. Taken together, our findings uncover a new role of TSA1 in preventing the overproduction of dNTPs, which is a root cause of genome instability.     

The conserved Tpk1 regulates non-homologous end joining double-strand break repair by phosphorylation of Nej1, a homolog of the human XLF

The yeast cyclic AMP-dependent protein kinase A (PKA) is a ubiquitous serine–threonine kinase, encompassing three catalytic (Tpk1–3) and one regulatory (Bcy1) subunits. Evidence suggests PKA involvement in DNA damage checkpoint response, but how DNA repair pathways are regulated by PKA subunits remains inconclusive. Here, we report that deleting the tpk1 catalytic subunit reduces non-homologous end joining (NHEJ) efficiency, whereas tpk2-3 and bcy1 deletion does not. Epistatic analyses revealed that tpk1, as well as the DNA damage checkpoint kinase (dun1) and NHEJ factor (nej1), co-function in the same pathway, and parallel to the NHEJ factor yku80. Chromatin immunoprecipitation and resection data suggest that tpk1 deletion influences repair protein recruitments and DNA resection. Further, we show that Tpk1 phosphorylation of Nej1 at S298 (a Dun1 phosphosite) is indispensable for NHEJ repair and nuclear targeting of Nej1 and its binding partner Lif1. In mammalian cells, loss of PRKACB (human homolog of Tpk1) also reduced NHEJ efficiency, and similarly, PRKACB was found to phosphorylate XLF (a Nej1 human homolog) at S263, a corresponding residue of the yeast Nej1 S298. Together, our results uncover a new and conserved mechanism for Tpk1 and PRKACB in phosphorylating Nej1 (or XLF), which is critically required for NHEJ repair.     

Antagonism of Chk1 signaling in the G2 DNA damage checkpoint by dominant alleles of Cdr1

Activation of the Chk1 protein kinase by DNA damage enforces a checkpoint that maintains Cdc2 in its inactive, tyrosine-15 (Y15) phosphorylated state. Chk1 downregulates the Cdc25 phosphatases and concomitantly upregulates the Wee1 kinases that control the phosphorylation of Cdc2. Overproduction of Chk1 causes G(2) arrest/delay independently of DNA damage and upstream checkpoint genes. We utilized this to screen fission yeast for mutations that alter sensitivity to Chk1 signaling. We describe three dominant-negative alleles of cdr1, which render cells supersensitive to Chk1 levels, and suppress the checkpoint defects of chk1Delta cells. Cdr1 encodes a protein kinase previously identified as a negative regulator of Wee1 activity in response to limited nutrition, but Cdr1 has not previously been linked to checkpoint signaling. Overproduction of Cdr1 promotes checkpoint defects and exacerbates the defective response to DNA damage of cells lacking Chk1. We conclude that regulation of Wee1 by Cdr1 and possibly by related kinases is an important antagonist of Chk1 signaling and represents a novel negative regulation of cell cycle arrest promoted by this checkpoint.     

DNA methylation inhibitor 5-Aza-2'-deoxycytidine induces reversible genome-wide DNA damage that is distinctly influenced by DNA methyltransferases 1 and 3B

Genome-wide DNA methylation patterns are frequently deregulated in cancer. There is considerable interest in targeting the methylation machinery in tumor cells using nucleoside analogs of cytosine, such as 5-aza-2′-deoxycytidine (5-azadC). 5-azadC exerts its antitumor effects by reactivation of aberrantly hypermethylated growth regulatory genes and cytoxicity resulting from DNA damage. We sought to better characterize the DNA damage response of tumor cells to 5-azadC and the role of DNA methyltransferases 1 and 3B (DNMT1 and DNMT3B, respectively) in modulating this process. We demonstrate that 5-azadC treatment results in growth inhibition and G₂ arrest—hallmarks of a DNA damage response. 5-azadC treatment led to formation of DNA double-strand breaks, as monitored by formation of γ-H2AX foci and comet assay, in an ATM (ataxia-telangiectasia mutated)-dependent manner, and this damage was repaired following drug removal. Further analysis revealed activation of key strand break repair proteins including ATM, ATR (ATM-Rad3-related), checkpoint kinase 1 (CHK1), BRCA1, NBS1, and RAD51 by Western blotting and immunofluorescence. Significantly, DNMT1-deficient cells demonstrated profound defects in these responses, including complete lack of γ-H2AX induction and blunted p53 and CHK1 activation, while DNMT3B-deficient cells generally showed mild defects. We identified a novel interaction between DNMT1 and checkpoint kinase CHK1 and showed that the defective damage response in DNMT1-deficient cells is at least in part due to altered CHK1 subcellular localization. This study therefore greatly enhances our understanding of the mechanisms underlying 5-azadC cytotoxicity and reveals novel functions for DNMT1 as a component of the cellular response to DNA damage, which may help optimize patient responses to this agent in the future.     

Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation

The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.     

Checkpoint-Dependent Regulation of Origin Firing and Replication Fork Movement in Response to DNA Damage in Fission Yeast

To elucidate the checkpoint mechanism responsible for slowing passage through S phase when fission yeast cells are treated with the DNA-damaging agent methyl methanesulfonate (MMS), we carried out two-dimensional gel analyses of replication intermediates in cells synchronized by cdc10 block (in G₁) followed by release into synchronous S phase. The results indicated that under these conditions early-firing centromeric origins were partially delayed but late-firing telomeric origins were not delayed. Replication intermediates persisted in MMS-treated cells, suggesting that replication fork movement was inhibited. These effects were dependent on the Cds1 checkpoint kinase and were abolished in cells overexpressing the Cdc25 phosphatase, suggesting a role for the Cdc2 cyclin-dependent kinase. We conclude that both partial inhibition of the firing of a subset of origins and inhibition of replication fork movement contribute to the slowing of S phase in MMS-treated fission yeast cells.In response to low levels of the DNA-alkylating agent methyl methanesulfonate (MMS), wild-type yeast cells slow their progression through S phase, while cells lacking the appropriate upstream checkpoint kinase (Mec1 in the budding yeast Saccharomyces cerevisiae; Rad3 in the fission yeast Schizosaccharomyces pombe) or the appropriate downstream checkpoint kinase (Rad53 in budding yeast, Cds1 in fission yeast) fail to do so. Other DNA-damaging agents also cause a checkpoint-dependent slowing of S phase, in vertebrates as well as in yeasts. This slowing of S phase in response to DNA damage is sometimes called the “intra-S-phase” checkpoint (3, 6, 22, 23, 26, 28, 36, 37, 45, 53). Here we shall refer to it as the “S-phase damage” checkpoint.Prior to this report, the downstream portions of the checkpoint pathway(s) that slow S phase in response to DNA damage in fission yeast were unclear. However, the upstream portions of these pathways in fission yeast and other organisms have been partially elucidated, and downstream mechanisms in other organisms have been partially clarified. In all studied systems, upon detection of DNA damage in S phase, checkpoint proteins initiate a phosphorylation cascade that ultimately leads to slowing of replication. Upstream signaling in these systems involves the activation of one or more of the phosphatidylinositol-3-kinase-like protein kinases (PIK kinases; ATR and/or ATM in humans, Mec1 and/or Tel1 in budding yeast, and Rad3 in fission yeast). The activated PIK kinases then phosphorylate several proteins, including certain Ser/Thr kinases (Chk1 and/or Chk2 in humans, Rad53 in budding yeast, and Cds1 in fission yeast). These kinases, in turn, phosphorylate other substrates that, directly or indirectly, mediate the slowing of S phase (reviewed in reference 3).In budding yeast, two different mechanisms were shown to slow S phase upon DNA damage by MMS. Of these, one mechanism, inhibition of late-firing origins, depended on the Mec1-Rad53 checkpoint pathway (45, 53), while the other mechanism, inhibition of replication forks, appeared to be a direct consequence of DNA damage rather than a result of checkpoint activation (53). Tercero and Diffley (53) found that, in MMS-treated cells with mutations in the RAD53 gene, unregulated origin firing compensated for checkpoint-independent replication fork slowing, thus permitting a relatively normal overall rate of DNA synthesis. The mechanism by which the Rad53 protein modulates late origin activity is not yet clear, but one possibility is inhibition (by Rad53-catalyzed phosphorylation) of Dbf4, the regulatory subunit of the Cdc7-Dbf4 kinase, which is essential for initiation of replication (7, 8, 14, 55).In vertebrates, at least three different pathways have been shown to contribute to the slowing of S phase after DNA damage. In some cases checkpoint-mediated phosphorylation of Dbf4 inhibits progression through S phase by downregulating origin firing (7, 14), as may take place in budding yeast. In other cases, checkpoint-mediated phosphorylation leads to inhibition and destruction of the protein phosphatase Cdc25A, which is an activator of Cdk2. Cdk2 is the S-phase-specific cyclin-dependent kinase. Cdk2 activity is crucial for initiation of DNA replication and is modulated by inhibitory phosphorylation at Tyr-15. Cdc25A activates Cdk2 by dephosphorylating Tyr-15. Thus, when Cdc25A is phosphorylated by checkpoint kinases after DNA damage and subsequently destroyed, Cdk2 can no longer promote initiation of DNA replication (9, 27). The third mechanism by which vertebrate cells can slow progression through S phase is inhibition of replication fork movement. In vertebrate cells, slowing of replication forks in response to DNA damage is frequently checkpoint dependent; in contrast, in budding yeast, such slowing appeared to be checkpoint independent. In the tested cases, fork slowing has proved to be dependent on the PIK kinase ATR (homologous to budding yeast Mec1 and fission yeast Rad3) and on the Ser/Thr kinase Chk1 (a functional analogue of budding yeast''s Rad53 and fission yeast''s Cds1). In each of these cases, the checkpoint response to DNA damage led to inhibition of origin firing as well as to inhibition of replication fork movement (42, 44, 54). The precise mechanism leading to slowing of replication fork movement has not been fully worked out, but the mechanism appears to involve interactions between Chk1 and the proteins Tim and Tipin (54), whose yeast homologues (Swi1 and Swi3 in fission yeast, Tof1 and Csm3 in budding yeast) form a “replication fork protection complex” that is associated with replication forks (19, 33).Although it is clear that slowing of S phase in response to MMS-induced DNA damage in fission yeast requires both the Rad3 and Cds1 kinases, the pathways operating downstream of Cds1 have been uncertain. We obtained results indicating that Cdc25, which was already known to be a target of Cds1 in hydroxyurea (HU)-treated cells, is also a target of Cds1 in MMS-treated cells, because both overproduction of Cdc25 and conversion of Tyr-15 on Cdc2 (the major cyclin-dependent kinase of fission yeast; also known as Cdk1) to a nonphosphorylatable residue (Cdc2-Y15F; this mutation rendered Cdc2 constitutively active) were sufficient to prevent MMS-induced slowing of S phase (23). We concluded that, in fission yeast, the Rad3→Cds1⊣Cdc25→Cdc2 pathway forms a checkpoint signaling module very similar to the corresponding one of vertebrates. However, Kommajosyula and Rhind were not able to repeat our observations regarding the roles of Cdc25 and Cdc2 (22), so the relevance of Cdc25 and Cdc2 to checkpoint-induced slowing of S phase in fission yeast has remained uncertain until now. In addition, whether S phase in MMS-treated fission yeast cells is slowed by inhibition of origin firing, by reduction in rate of fork movement, or by a combination of these has been equally unclear.In order to resolve these issues, we initiated the series of experiments reported in this paper. To measure the rate of progression through S phase, we followed S phase by flow cytometry and by two-dimensional (2D) gel electrophoresis in cells released from a G₁ block (achieved by incubating cells bearing a cdc10 temperature-sensitive mutation at the restrictive temperature, then releasing to the permissive temperature [21, 23]). We found that, in MMS-treated, checkpoint-competent cells, the firing of early origins near centromeres was partially delayed but that the firing of late origins near telomeres was unaffected. Furthermore, the lifetimes of replication intermediates (RIs) were prolonged, consistent with slowing of replication forks. These effects were completely abrogated both in cells lacking the Cds1 kinase and in cells overproducing the Cdc25 phosphatase, showing that these effects were checkpoint dependent and that the relevant checkpoint pathway probably involved inhibition of Cdc25.