Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation

Abstract

Two classes of kinases, CDK and DDK, facilitate the initiation of DNA replication in S phase. In two studies, the Diffley and Toczyski labs show that when damage is sensed, another kinase — the checkpoint kinase Rad53 — halts replication by inhibiting both the CDK and DDK pathways through the phosphorylation of Sld3 and Dbf4, respectively. These results reveal that regulation of the firing of origins is the means by which the intra-S checkpoint slows S phase. Two classes of enzyme — cyclin-dependent kinases (CDK) and Dbf4-dependent kinase (DDK) — facilitate the initiation of DNA replication in eukaryotes. It is now shown that, when DNA damage is sensed, another kinase, Rad53, halts the firing of late replication origins by inhibiting both the CDK and the DDK pathways. Rad53 acts on DDK directly by inhibiting Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate Sld3. The initiation of eukaryotic DNA replication is regulated by three protein kinase classes: cyclin-dependent kinases (CDK), Dbf4-dependent kinase (DDK) and the DNA damage checkpoint kinases1. CDK phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb11 (refs 2–4), whereas DDK acts by phosphorylating subunits of the Mcm2-7 helicase5. CDK has an additional role in replication by preventing the re-loading of Mcm2-7 during the S, G2 and M phases6, thus preventing origin re-firing and re-replication. During the G1 phase, both CDK and DDK are downregulated, which allows origin licensing and prevents premature replication initiation3. Origin firing is also inhibited during the S phase when DNA damage or replication fork stalling activates the checkpoint kinases7,8,9,10. Here we show that, analogous to the situation in the G1 phase, the Saccharomyces cerevisiae checkpoint kinase Rad53 inhibits both CDK- and DDK-dependent pathways, which acts redundantly to block further origin firing. Rad53 acts on DDK directly by phosphorylating Dbf4, whereas the CDK pathway is blocked by Rad53-mediated phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during the S phase in the presence of DNA damage, which is crucial to prevent re-loading of Mcm2-7 onto origins that have already fired6. Our results explain how checkpoints regulate origin firing and demonstrate that the slowing of S phase by the ‘intra-S checkpoint’ is primarily due to the inhibition of origin firing.