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Drf1-dependent Kinase Interacts with Claspin through a Conserved Protein Motif

Gold, Daniel A. and Dunphy, William G. (2010) Drf1-dependent Kinase Interacts with Claspin through a Conserved Protein Motif. Journal of Biological Chemistry, 285 (17). pp. 12638-12646. ISSN 0021-9258. PMCID PMC2857117.

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The Dbf4/Drf1-dependent kinase (DDK) is required for the initiation of DNA replication in eukaryotes. Another protein, Claspin, mediates the activation of a cellular checkpoint response to stalled replication forks and is also a regulator of replication. In this study, we found that DDK phosphorylates Claspin in vitro and forms a nuclear complex containing Cdc7, Drf1, and Claspin in Xenopus egg extracts. In addition, purified Claspin and DDK are capable of a direct in vitro interaction. We identified a conserved binding site on Claspin required for its interaction with DDK. This site corresponds to the first of two sequence repeats in the Chk1-binding domain of Claspin. Furthermore, we have established that two amino acids in this motif, Asp^(861) and Gln^(866), are essential for the interaction between Claspin and DDK. We found that mutant forms of Claspin incapable of interacting with DDK are still able to associate with and activate Chk1 in response to DNA replication blockages. However, Claspin-depleted egg extracts that have been reconstituted with these mutants of Claspin undergo DNA replication more slowly. These findings suggest that the interaction of DDK with Claspin mediates a checkpoint-independent function of Claspin related to DNA replication.

Item Type:Article
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URLURL TypeDescription DOIArticle CentralArticle
Gold, Daniel A.0000-0003-0135-4022
Dunphy, William G.0000-0001-7598-8939
Additional Information:© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Received for publication, October 19, 2009, and in revised form, February 18, 2010. Published, JBC Papers in Press, February 27, 2010. This work was supported, in whole or in part, by National Institutes of Health Grants GM043974 and GM070891 (to W. G. D.). We are grateful to our colleagues in the laboratory for advice and assistance throughout the course of this work and for comments on the manuscript. In addition, we thank Stephanie Yanow for preparing the baculovirus encoding His6-Drf1.
Funding AgencyGrant Number
Subject Keywords:Cell Cycle, Cell Division, Checkpoint Control, DNA Replication, Protein Kinases, Xenopus Egg Extract
Issue or Number:17
PubMed Central ID:PMC2857117
Record Number:CaltechAUTHORS:20100517-092639612
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Official Citation:Daniel A. Gold and William G. Dunphy Drf1-dependent Kinase Interacts with Claspin through a Conserved Protein Motif J. Biol. Chem. 2010 285: 12638-12646. First Published on February 27, 2010, doi:10.1074/jbc.M109.077370
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:18318
Deposited By: Tony Diaz
Deposited On:24 Jun 2010 20:51
Last Modified:09 Mar 2020 13:18

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