Interplay between S-cyclin-dependent kinase and Dbf4-dependent kinase in controlling DNA replication through phosphorylation of yeast Mcm4 N-terminal domain

Cyclin-dependent (CDK) and Dbf4-dependent (DDK) kinases trigger DNA replication in all eukaryotes, but how these kinases cooperate to regulate DNA synthesis is largely unknown. Here, we show that budding yeast Mcm4 is phosphorylated in vivo during S phase in a manner dependent on the presence of five CDK phosphoacceptor residues within the N-terminal domain of Mcm4. Mutation to alanine of these five sites (mcm4-5A) abolishes phosphorylation and decreases replication origin firing efficiency at 22 degrees C. Surprisingly, the loss of function mcm4-5A mutation confers cold and hydroxyurea sensitivity to DDK gain of function conditions (mcm5/bob1 mutation or DDK overexpression), implying that phosphorylation of Mcm4 by CDK somehow counteracts negative effects produced by ectopic DDK activation. Deletion of the S phase cyclins Clb5,6 is synthetic lethal with mcm4-5A and mimics its effects on DDK up mutants. Furthermore, we find that Clb5 expressed late in the cell cycle can still suppress the lethality of clb5,6 Delta bob1 cells, whereas mitotic cyclins Clb2, 3, or 4 expressed early cannot. We propose that the N-terminal extension of eukaryotic Mcm4 integrates regulatory inputs from S-CDK and DDK, which may play an important role for the proper assembly or stabilization of replisome-progression complexes.