Hierarchy of S-phase-promoting factors: yeast Dbf4-Cdc7 kinase requires prior S-phase cyclin-dependent kinase activation.
In all eukaryotes, the initiation of DNA synthesis requires the formation of prereplicative complexes (pre-RCs) on replication origins, followed by their activation by two S-T protein kinases, an S-phase cyclin-dependent kinase (S-CDK) and a homologue of yeast Dbf4-Cdc7 kinase (Dbf4p-dependent kinase [DDK]). Here, we show that yeast DDK activity is ... cell cycle regulated, though less tightly than that of the S-CDK Clb5-Cdk1, and peaks during S phase in correlation with Dbf4p levels. Dbf4p is short-lived throughout the cell cycle, but its instability is accentuated during G(1) by the anaphase-promoting complex. Downregulating DDK activity is physiologically important, as joint Cdc7p and Dbf4p overexpression is lethal. Because pre-RC formation is a highly ordered process, we asked whether S-CDK and DDK need also to function in a specific order for the firing of origins. We found that both kinases are activated independently, but we show that DDK can perform its function for DNA replication only after S-CDKs have been activated. Cdc45p, a protein needed for initiation, binds tightly to chromatin only after S-CDK activation (L. Zou and B. Stillman, Science 280:593-596, 1998). We show that Cdc45p is phosphorylated by DDK in vitro, suggesting that it might be one of DDK's critical substrates after S-CDK activation. Linking the origin-bound DDK to the tightly regulated S-CDK in a dependent sequence of events may ensure that DNA replication initiates only at the right time and place.
Mesh Terms:
Blotting, Northern, Blotting, Western, CDC2 Protein Kinase, Carrier Proteins, Cell Cycle, Cell Cycle Proteins, Cyclin B, Cyclin-Dependent Kinases, DNA-Binding Proteins, Enzyme Activation, Fungal Proteins, Intracellular Signaling Peptides and Proteins, Nuclear Proteins, Phosphorylation, Protein Kinases, Protein-Serine-Threonine Kinases, RNA Processing, Post-Transcriptional, S Phase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Blotting, Northern, Blotting, Western, CDC2 Protein Kinase, Carrier Proteins, Cell Cycle, Cell Cycle Proteins, Cyclin B, Cyclin-Dependent Kinases, DNA-Binding Proteins, Enzyme Activation, Fungal Proteins, Intracellular Signaling Peptides and Proteins, Nuclear Proteins, Phosphorylation, Protein Kinases, Protein-Serine-Threonine Kinases, RNA Processing, Post-Transcriptional, S Phase, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Mol. Cell. Biol.
Date: Jun. 01, 2000
PubMed ID: 10805723
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