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 kinases1. CDK phosphorylation of two key initiation factors, Sld2 and Sld3, promotes essential interactions with Dpb112-4 whilst DDK acts by phosphorylating subunits of the Mcm2-7 helicase5. CDK plays an additional role in replication by preventing the re-loading of Mcm2-7 during S, G2 and M phases6, thus preventing origin re-firing and re-replication. During G1-phase, both CDK and DDK are down-regulated, which allows origin licensing and prevents premature replication initiation3. Origin firing is also inhibited during S-phase when DNA damage or replication fork stalling activates the checkpoint kinases7-10. Here we show that, analogous to the situation in G1 phase, the 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 phosphorylation of the downstream CDK substrate, Sld3. This allows CDK to remain active during S phase in the presence of DNA damage, which is crucial to prevent reloading 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.