title: Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway.
abstract: Eukaryotic cells coordinate chromosome duplication by assembly of protein complexes at origins of DNA replication and by activation of cyclin-dependent kinase and Cdc7p-Dbf4p kinase. We show in Saccharomyces cerevisiae that although Cdc7p levels are constant during the cell division cycle, Dbf4p and Cdc7p-Dbf4p kinase activity fluctuate. Dbf4p binds to chromatin near the G(1)/S-phase boundary well after binding of the minichromosome maintenance (Mcm) proteins, and it is stabilized at the non-permissive temperature in mutants of the anaphase-promoting complex, suggesting that Dbf4p is targeted for destruction by ubiquitin-mediated proteolysis. Arresting cells with hydroxyurea (HU) or with mutations in genes encoding DNA replication proteins results in a more stable, hyper-phosphorylated form of Dbf4p and an attenuated kinase activity. The Dbf4p phosphorylation in response to HU is RAD53 dependent. This suggests that an S-phase checkpoint function regulates Cdc7p-Dbf4p kinase activity. Cdc7p may also play a role in adapting from the checkpoint response since deletion of CDC7 results in HU hypersensitivity. Recombinant Cdc7p-Dbf4p kinase was purified and both subunits were autophosphorylated. Cdc7p-Dbf4p efficiently phosphorylates several proteins that are required for the initiation of DNA replication, including five of the six Mcm proteins and the p180 subunit of DNA polymerase alpha-primase.
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
authors.forename: M
authors.initials: M
authors.lastname: Weinreich
authors.forename: B
authors.initials: B
authors.lastname: Stillman
mesh_terms: D018797:Cell Cycle Proteins / Q000302:isolation & purification / Q000378:metabolism*; D064447:Checkpoint Kinase 2; D002843:Chromatin / Q000378:metabolism*; D004256:DNA Polymerase I / Q000378:metabolism; D019915:DNA Primase / Q000378:metabolism; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017491:Genes, APC; D006918:Hydroxyurea / Q000494:pharmacology; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
chemical_list: D018797:Cell Cycle Proteins; D002843:Chromatin; C088096:Dbf4 protein, S cerevisiae; D005656:Fungal Proteins; D010750:Phosphoproteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C489404:CDC7 protein, S cerevisiae; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D019915:DNA Primase; C116932:DNA polymerase alpha-primase; D004256:DNA Polymerase I; D006918:Hydroxyurea