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J. Biol. Chem., Vol. 282, Issue 49, 35910-35923, December 7, 2007

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Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair^*

From the Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016

The activity of human replication protein A (RPA) in DNA replication and repair is regulated by phosphorylation of the middle RPA2 subunit. It has previously been shown that up to nine different N-terminal residues are modified in vivo and in response to genotoxic stress. Using a novel antibody against phospho-Ser²⁹, a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser²⁹ was also seen in interphase cells following treatment with the DNA-damaging agent camptothecin, a rare example of stress stimulating the modification of a repair factor by cyclin-Cdk. RPA2 phosphorylation is regulated both in cis and trans. Cis-phosphorylation follows a preferred pathway. (That is, the initial modification of Ser³³ by ATR stimulates subsequent phosphorylation of Cdk sites Ser²³ and Ser²⁹). These events then facilitate modification of Thr²¹ and extreme N-terminal sites Ser⁴ and Ser⁸, probably by DNA-PK. Our data also indicate that the phosphorylation of one RPA molecule can influence the phosphorylation of other RPA molecules in trans. Cells in which endogenous RPA2 was "replaced" with a double S23A/S29A-RPA2 mutant were seen to have an abnormal cell cycle distribution both in normal and in stressed cells. Such cells also showed aberrant DNA damage-dependent RPA foci and had persistent staining of H2AX following DNA damage. Our data indicate that RPA phosphorylation facilitates chromosomal DNA repair. We postulate that the RPA phosphorylation pattern provides a means to regulate the DNA repair pathway utilized.

Received for publication, June 6, 2007 , and in revised form, October 9, 2007.

^* This work was supported by Department of Defense Breast Cancer Research Program Grant DAMD17-03-1-0299, National Institutes of Health Grant AI29963, and NCI, National Institutes of Health, Grant P30CA16087 (to the New York University Cancer Institute and Rita J. and Stanley Kaplan Comprehensive Cancer Center). 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 Biochemistry, New York University School of Medicine, 550 First Ave., New York, NY 10016. Tel.: 212-263-8453; Fax: 212-263-8166; E-mail: james.borowiec{at}med.nyu.edu.

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