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J. Biol. Chem., Vol. 284, Issue 1, 182-190, January 2, 2009

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ATR^Mec1 Phosphorylation-independent Activation of Chk1 in Vivo^*

Yinhuai Chen, Julie M. Caldwell¹, Elizabeth Pereira, Robert W. Baker, and Yolanda Sanchez²

From the Department of Pharmacology and Toxicology, Norris Cotton Cancer Center, Dartmouth Medical School, Hanover, New Hampshire 03755 and Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524

The conserved protein kinase Chk1 is a player in the defense against DNA damage and replication blocks. The current model is that after DNA damage or replication blocks, ATR^Mec1 phosphorylates Chk1 on the non-catalytic C-terminal domain. However, the mechanism of activation of Chk1 and the function of the Chk1 C terminus in vivo remains largely unknown. In this study we used an in vivo assay to examine the role of the C terminus of Chk1 in the response to DNA damage and replication blocks. The conserved ATR^Mec1 phosphorylation sites were essential for the checkpoint response to DNA damage and replication blocks in vivo; that is, that mutation of the sites caused lethality when DNA replication was stalled by hydroxyurea. Despite this, loss of the ATR^Mec1 phosphorylation sites did not change the kinase activity of Chk1 in vitro. Furthermore, a single amino acid substitution at an invariant leucine in a conserved domain of the non-catalytic C terminus restored viability to cells expressing the ATR^Mec1 phosphorylation site-mutated protein and relieved the requirement of an upstream mediator for Chk1 activation. Our findings show that a single amino acid substitution in the C terminus, which could lead to an allosteric change in Chk1, allows it to bypass the requirement of the conserved ATR^Mec1 phosphorylation sites for checkpoint function.

Received for publication, August 22, 2008 , and in revised form, November 3, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants P30 ES06096 and U01 ES11038 (NIEHS) and RO1 CA84463 (NCI) (to Y. S.). This work was also supported by Pew Scholars Program in the Biomedical Sciences (to Y. S.). 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 on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ A Ryan Fellow. Present address: Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45220-3039.

² To whom correspondence should be addressed: 7650 Remsen, Hanover, NH 03755. Fax: 603-650-1129; E-mail: Yolanda.Sanchez{at}Dartmouth.edu.

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