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J. Biol. Chem., Vol. 282, Issue 2, 986-995, January 12, 2007

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Mechanism of Dun1 Activation by Rad53 Phosphorylation in Saccharomyces cerevisiae^*

Sheng-hong Chen, Marcus B. Smolka, and Huilin Zhou^¶1

From the Division of Biological Sciences, Ludwig Institute for Cancer Research, and the ^¶Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093-0653

Despite extensive studies, the molecular mechanism of DNA damage checkpoint activation remains incompletely understood. To better dissect this mechanism, we developed an activity-based assay for Dun1, a downstream DNA damage check-point kinase in yeast, using its physiological substrate Sml1. Using this assay, we confirmed the genetic basis of Dun1 activation. Rad53 was found to be directly responsible for Dun1 activation. We reconstituted the activation of Dun1 by Rad53 and found that phosphorylation of Thr-380 in the activation loop of Dun1 by Rad53 is responsible for Dun1 activation. Interestingly, phosphorylation of the evolutionarily conserved Thr-354 in the activation loop of Rad53 is also important for the regulation of Rad53 activity. Thus, this conserved mode of activation loop phosphorylation appears to be a general mechanism for the activation of Chk2 family kinases.

Received for publication, October 2, 2006 , and in revised form, November 8, 2006.

^* This work was supported by a K22 Faculty Transition Grant HG002604 from National Human Genome Research Institute and additional research support from the Ludwig Institute for Cancer Research (to H. Z.). 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 Tables S1 and S2.

¹ To whom correspondence should be addressed: Ludwig Institute for Cancer Research, University of California San Diego, 9500 Gilman Dr., CMM-East, Rm. 3050, La Jolla, CA 92093-0653. Tel.: 858-552-4920 (ext. 7808); Fax: 858-534-7750; E-mail: huzhou{at}ucsd.edu.

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