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J. Biol. Chem., Vol. 279, Issue 48, 49599-49608, November 26, 2004

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Claspin and the Activated Form of ATR-ATRIP Collaborate in the Activation of Chk1^*

Akiko Kumagai, Soo-Mi Kim, and William G. Dunphy

From the Division of Biology, California Institute of Technology, Pasadena, California 91125

Claspin is necessary for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. ATR possesses a regulatory partner called ATRIP. We have studied the respective roles of ATR-ATRIP and Claspin in the activation of Chk1. ATR-ATRIP bound well to various DNA templates in Xenopus egg extracts. ATR-ATRIP bound to a single-stranded DNA template was weakly active. By contrast, the ATR-ATRIP complex on a DNA template containing both single- and double-stranded regions displayed a large increase in kinase activity. This observation suggests that ATR-ATRIP normally undergoes activation upon association with specific nucleic acid structures at DNA replication forks. Without Claspin, activated ATR-ATRIP phosphorylated Chk1 weakly in a cell-free reaction. The addition of Claspin to this reaction strongly stimulated the phosphorylation of Chk1 by ATR-ATRIP. Claspin also induced significant autophosphorylation of Chk1 in the absence of ATR-ATRIP. Taken together, these results indicate that the checkpoint-dependent phosphorylation of Chk1 is a multistep process involving activation of the ATR-ATRIP complex at replication forks and presentation of Chk1 to this complex by Claspin.

Received for publication, July 23, 2004 , and in revised form, September 13, 2004.

^* This work was supported in part by National Institutes of Health Grants GM043974 and GM070891. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY644453.

To whom correspondence should be addressed: Div. of Biology, 216-76, California Inst. of Technology, 1200 E. California Blvd., Pasadena, CA 91125. Tel.: 626-395-8433; Fax: 626-795-7563; E-mail: dunphy{at}cco.caltech.edu.

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