Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication

The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replicat ion in Saccharomyces cerevisiae, Previously, we identified a human Cdc7 hom olog, HsCdc7. In this study, we report the identification of a human Dbf4 h omolog, HsDbf4. We show that HsDbf4 binds to HsCdc7 and activates HsCdc7 ki nase activity when HsDbf4 and HsCdc7 are coexpressed in insect and mammalia n cells. HsDbf4 protein levels are regulated during the cell cycle with a p attern that matches that of HsCdc7 protein kinase activity. They are low in G(1), increase during G(1)-S, and remain high during S and G(2)-M. Purifie d baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 s ubunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro, Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo P-32-labeled MCM2 from HeLa cell s reveals that several major tryptic phosphopeptides of MCM2 comigrate with those of MCM2 phosphorylated by HsCdc7-HsDbf4 in vitro, suggesting that MC M2 is a physiological HsCdc7-HsDbf4 substrate, Immunoneutralization of HsCd c7HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cel ls blocks initiation of DNA replication. These results indicate that the Hs Cdc7HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells.