Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway

Weinreich, M., Stillman, B. (October 1999) Cdc7p-Dbf4p kinase binds to chromatin during S phase and is regulated by both the APC and the RAD53 checkpoint pathway. EMBO Journal, 18 (19). pp. 5334-46. ISSN 02614189 (ISSN)

DOI: 10.1093/emboj/18.19.5334


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.

Item Type: Paper
Uncontrolled Keywords: Cell Cycle Proteins isolation & purification metabolism Chromatin metabolism DNA Polymerase I metabolism DNA Primase metabolism Fungal Proteins isolation & purification metabolism Genes, APC Hydroxyurea pharmacology Phosphoproteins metabolism Protein Binding Protein Kinases genetics Protein-Serine-Threonine Kinases isolation & purification metabolism S Phase drug effects Saccharomyces cerevisiae Proteins Substrate Specificity
Subjects: organism description > yeast > Saccharomyces
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > Chromatin dynamics
CSHL Authors:
Communities: CSHL labs > Stillman lab
Highlight: Stillman, Bruce W.
Depositing User: CSHL Librarian
Date: 1 October 1999
Date Deposited: 21 Feb 2012 16:56
Last Modified: 20 Jun 2017 19:22
PMCID: PMC1171603
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