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J. Biol. Chem., Vol. 281, Issue 51, 39517-39533, December 22, 2006

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RPA2 Is a Direct Downstream Target for ATR to Regulate the S-phase Checkpoint^*

Erin Olson, Christian J. Nievera, Vitaly Klimovich, Ellen Fanning, and Xiaohua Wu¹

From the Department of Molecular and Experimental Medicine, The Scripps Research Institute, La, Jolla, California 92037 and the Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee 37232

Upon DNA damage, replication is inhibited by the S-phase checkpoint. ATR (ataxia telangiectasia mutated- and Rad3-related) is specifically involved in the inhibition of replicon initiation when cells are treated with DNA damage-inducing agents that stall replication forks, but the mechanism by which it acts to prevent replication is not yet fully understood. We observed that RPA2 is phosphorylated on chromatin in an ATR-dependent manner when replication forks are stalled. Mutation of the ATR-dependent phosphorylation sites in RPA2 leads to a defect in the down-regulation of DNA synthesis following treatment with UV radiation, although ATR activation is not affected. Threonine 21 and serine 33, two residues among several phosphorylation sites in the amino terminus of RPA2, are specifically required for the UV-induced, ATR-mediated inhibition of DNA replication. RPA2 mutant alleles containing phospho-mimetic mutations at ATR-dependent phosphorylation sites have an impaired ability to associate with replication centers, indicating that ATR phosphorylation of RPA2 directly affects the replication function of RPA. Our studies suggest that in response to UV-induced DNA damage, ATR rapidly phosphorylates RPA2, disrupting its association with replication centers in the S-phase and contributing to the inhibition of DNA replication.

Received for publication, May 30, 2006 , and in revised form, October 10, 2006.

^* This work was supported by National Institutes of Health Grant CA102361, Ellison Medical Foundation New Scholar Award AG-NS-0251-04 (to X. W.), and National Institutes of Health Training Grant 5 T32 DK007022 (to E. O.). 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. 1-3.

¹ To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-7910; Fax: 858-784-7978; E-mail: xiaohwu{at}scripps.edu.

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