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)
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.
| 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 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/25135 |
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