Regulation of the replication initiator protein p65cdc18 by CDK phosphorylation

Cyclin-dependent kinases (CDKs) promote the initiation of DNA replication and prevent reinitiation before mitosis, presumably through phosphorylation of key substrates at origins of replication. In fission yeast, the p65^cdc18 protein is required to initiate DNA replication and interacts with the origin recognition complex (ORC) and the p34^cdc2 CDK. Here we report that p65^cdc18 becomes highly phosphorylated as cells undergo the G₁→S phase transition. This modification is dependent on p34^cdc2 protein kinase activity, as well as six consensus CDK phosphorylation sites within the p65^cdc18 polypeptide. Genetic interactions between cdc18⁺ and the S-phase cyclin cig2⁺ suggest that CDK-dependent phosphorylation antagonizes cdc18⁺ function in vivo. Using site-directed mutagenesis, we show that phosphorylation at CDK consensus sites directly targets p65^cdc18 for rapid degradation and inhibits its replication activity, as strong expression of a constitutively hypophosphorylated mutant form of p65^cdc18 results in large amounts of DNA over-replication in vivo. Furthermore, the over-replication phenotype produced by this mutant p65^cdc18 is resistant to increased mitotic cyclin/CDK activity, a known inhibitor of over-replication. Therefore, p65^cdc18 is the first example of a cellular initiation factor directly regulated in vivo by CDK-dependent phosphorylation and proteolysis. Regulation of p65^cdc18 by CDK phosphorylation is likely to contribute to the CDK-driven “replication switch” that restricts initiation at eukaryotic origins to once per cell cycle.