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

Abstract

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 (RPA2_D) or alanine (RPA2_A). Although RPA2_D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2_D mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2_D 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 RPA2_D. In contrast, under DNA damage or replication stress conditions, RPA2_D, like RPA2_A and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with γ-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.

We thank Kyung Kim and Diana Dimitrova for helpful discussions during the course of these experiments, Kristine Carta for expert technical assistance, and John Hirsch for assistance with FACS analysis.

J.A.B. was supported by NIH grant AI29963, DOD Breast Cancer Research Program DAMD17-03-1-0299, Philip Morris grant 15-B0001-42171, and the NYU Cancer Institute and the Rita J. and Stanley Kaplan Comprehensive Cancer Center (NCI P30CA16087). M.S.W. was supported by NIH grant GM44721.