Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway.
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.
Weinreich
M
M
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Stillman
B
B
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
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