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.