Abstract
The Cdc7-Dbf4 complex is a conserved serine/threonine protein kinase essential for the initiation of eukaryotic DNA replication. Although an mcm5-bob1 mutation bypasses lethality conferred by mutations in CDC7 or DBF4, the Deltacdc7 mcm5-bob1 mutant is sensitive to hydroxyurea (HU), which induces replication stress. To elucidate the reasons for HU sensitivity conferred by deletion of CDC7, we examined the role of Cdc7-Dbf4 in the replication checkpoint. We found that in Cdc7-Dbf4-deficient cells exposed to replication stress, Rad53 remains in a hypophosphorylated form, anaphase spindle is elongated, and checkpoint-specific transcription is not induced. The hypophosphorylated Rad53 exhibits a low autophosphorylation activity, and recombinant Cdc7-Dbf4 phosphorylates Rad53 in vitro. These results suggest that Cdc7-Dbf4 is required for full activation of Rad53 in response to replication stress.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Anaphase
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Blotting, Western
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Cell Cycle Proteins / physiology*
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Checkpoint Kinase 2
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DNA Replication*
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DNA, Fungal / physiology*
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Gene Deletion
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Gene Expression Regulation, Fungal
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Hydroxyurea / pharmacology
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Phosphorylation
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Protein Serine-Threonine Kinases / genetics
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Protein Serine-Threonine Kinases / metabolism*
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Protein Serine-Threonine Kinases / physiology*
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S Phase / physiology
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / growth & development
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Saccharomyces cerevisiae Proteins / physiology*
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beta-Galactosidase / metabolism
Substances
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Cell Cycle Proteins
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DNA, Fungal
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Dbf4 protein, S cerevisiae
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Saccharomyces cerevisiae Proteins
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CDC7 protein, S cerevisiae
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Checkpoint Kinase 2
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Protein Serine-Threonine Kinases
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RAD53 protein, S cerevisiae
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beta-Galactosidase
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Hydroxyurea