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.
Nougarède
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Institute of Molecular Genetics, CNRS UMR 5535 and Université Montpellier II, F-34293 Montpellier cedex 5, France. schwob@jones.igm.cnrs-mop.fr
Della Seta
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Zarzov
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Schwob
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D015152:Blotting, Northern; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism*; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins
C102209:CDC45 protein, S cerevisiae; C076558:CLB5 protein, S cerevisiae; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D019926:Cyclin B; D004268:DNA-Binding Proteins; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C489404:CDC7 protein, S cerevisiae; C064979:IME2 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D018844:Cyclin-Dependent Kinases