文档介绍：The EMBO Journal –5713, 1999 Mammalian Cdc7–Dbf4 protein plex is essential for initiation of DNA replication Wei Jiang 1,2, David McDonald, Thomas and Tony Hunter The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA 2Present address: Department of Biochemistry, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA 1Corresponding author e-mail: ******@ The Cdc7–Dbf4 kinase is essential for regulating initi- ation of DNA replication in haromyces cerevisiae . Previously, we identi?ed a human Cdc7 homolog, HsCdc7. In this study, we report the identi?cation of a human Dbf4 homolog, HsDbf4. We show that HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity whenHsDbf4 and HsCdc7are coexpressed in insect and mammalian cells. HsDbf4 protein levels are regulated during the cell cycle with a pattern 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. Puri?ed baculovirus-expressed HsCdc7– HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) plex isolated by immunoprecipitation with MCM7 antibodies in vitro . Two-dimensional tryptic phospho- peptide-mapping analysis of in vivo 32P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of igrate with those of MCM2 phosphorylated by HsCdc7–HsDbf4 in vitro , suggesting that MCM2 is a physiological HsCdc7– HsDbf4 substrate. Immunoneutralization of HsCdc7– HsDbf4 activity by microinjection of anti-HsCdc7 antibodies into HeLa cells blocks initiation of DNA replication. These results indicate that the HsCdc7– HsDbf4 kinase is directly involved in regulating the initiation of DNA replication by targeting MCM2 protein in mammalian cells. Keywords : DNA replication/HsCdc7/HsDbf4/kinase/ MCM Introduction Eukaryotic chromosomal replication is a tightly regulated process, which must be strictly coordinated with