Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication.
The Cdc7-Dbf4 kinase is essential for regulating initiation of DNA replication in Saccharomyces cerevisiae. Previously, we identified a human Cdc7 homolog, HsCdc7. In this study, we report the identification of a human Dbf4 homolog, HsDbf4. We show that HsDbf4 binds to HsCdc7 and activates HsCdc7 kinase activity when HsDbf4 and HsCdc7 are 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. Purified baculovirus-expressed HsCdc7-HsDbf4 selectively phosphorylates the MCM2 subunit of the minichromosome maintenance (MCM) protein complex isolated by immunoprecipitation with MCM7 antibodies in vitro. Two-dimensional tryptic phosphopeptide-mapping analysis of in vivo (32)P-labeled MCM2 from HeLa cells reveals that several major tryptic phosphopeptides of MCM2 comigrate 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.
Jiang
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The Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA. wjiang@salk.edu
McDonald
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Hope
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TJ
Hunter
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D000595:Amino Acid Sequence; D000818:Animals; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004261:DNA Replication / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D064111:Minichromosome Maintenance Complex Component 2; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C489404:CDC7 protein, S cerevisiae; C068619:CDC7 protein, human; C489403:Cdc7 protein, mouse; D017346:Protein Serine-Threonine Kinases; D064111:Minichromosome Maintenance Complex Component 2