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

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 kinases(1). 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 helicase(5). CDK has an additional role in replication by preventing the re-loading of Mcm2-7 during the S, G2 and M phases(6), 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 initiation(3). Origin firing is also inhibited during the S phase when DNA damage or replication fork stalling activates the checkpoint kinases(7-10). 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 fired(6). 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.