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Molecular and Cellular Biology, March 2004, p. 1930-1943, Vol. 24, No. 5
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.5.1930-1943.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

Vitaly M. Vassin,1 Marc S. Wold,2 and James A. Borowiec1*

Department of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, New York 10016,1 Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 522422

Received 11 August 2003/ Returned for modification 15 September 2003/ Accepted 24 November 2003

Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2D again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2D. In contrast, under DNA damage or replication stress conditions, RPA2D, like RPA2A and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with {gamma}-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.

* Corresponding author. Mailing address: Dept. of Biochemistry and New York University Cancer Institute, New York University School of Medicine, New York, NY 10016. Phone: (212) 263-8453. Fax: (212) 263-8166. E-mail: james.borowiec{at}med.nyu.edu.

Molecular and Cellular Biology, March 2004, p. 1930-1943, Vol. 24, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.5.1930-1943.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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