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-Ser 29 , a moiety formed by cyclin-Cdk, we observed that RPA2 was phosphorylated during mitosis in nonstressed cells. Robust phosphorylation of Ser 29 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 Ser 33 by ATR stimulates subsequent phosphorylation of Cdk sites Ser 23 and Ser 29 ). These events then facilitate modification of Thr 21 and extreme N-terminal sites Ser 4 and Ser 8 , 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.Replication protein A (RPA) 2 is a heterotrimeric singlestranded DNA-binding factor that is critical for the "three Rs" of eukaryotic DNA enzymology: DNA replication, DNA recombination, and DNA repair (1, 2). For DNA replication, the study of cellular and viral model systems demonstrates that RPA is needed both for origin denaturation and replication elongation, in the latter case to facilitate the switch from DNA polymerase ␣ to DNA polymerase ␦ during Okazaki fragment synthesis (3). RPA acts in homologous recombination (HR) to stimulate DNA annealing using physical interactions with Rad52 (4 -7) and in HR-mediated DNA repair, probably employing specific interactions with BRCA2 (8, 9). RPA is a required factor in both the nucleotide excision (10, 11) and mismatch repair pathways (12, 13) and in somatic hypermutation (14). Because of these many roles, it is of significant interest to understand the mechanisms that regulate RPA activity.Of the ϳ70-kDa (RPA1), 30-kDa (RPA2), and 14-kDa (RPA3) subunits, human RPA is subject to extensive phosphorylation on RPA2 (2) and at one RPA1 site (15). The N-terminal 33 residues of RPA2 undergo both cell cycle-and stress-dependent phosphorylation on approximately nine sites (Fig. 1A), which are thought to exist in an unstructured conformation (16,17). Ser 23 and Ser 29 are constitutively modified during mitosis by cyclin B-Cdk1 (18, 19) and have been suggested to be partially modified beginni...

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 ␥-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.DNA-damaging stress leads to the inception of a variety of cellular responses that serve to minimize mutation and prevent genomic instability. In particular, the cell cycle checkpoint apparatus is activated to block S phase entry and, in those cells in the replicative phase, to both inhibit firing of late origins of DNA replication and avert the collapse of replication forks blocked by damage (3). The DNA repair machinery is mobilized in concert to repair lesions and to allow eventual restart of stalled replication forks. One factor that plays essential roles both during DNA replication and in the repair-and recombination-mediated recovery from damage is replication protein A (RPA), the eukaryotic single-stranded (ss) DNA-binding protein (27,52).RPA is a heterotrimeric protein consisting, in mammalian cells, of ϳ70-(RPA1), 30-(RPA2), and 14 (RPA3)-kDa subunits. During DNA replication, RPA acts at the fork, stabilizing ssDNA and facilitating nascent strand synthesis by the replicative DNA polymerases. Under DNA-damaging conditions, RPA-ssDNA complexes act to recruit and activate a key checkpoint mediator consisting of the ATR and ATRIP (ATRinteracting protein) protein-kinase complex (54). At DNA damage-dependent nuclear foci, RPA interacts with repair and recombination components to process double-strand DNA breaks and other lesions (19). RPA activity is regulated by various stress conditions. In particular, heat shock (12, 47, 48), exposure to UV radiation (9), and treatment with DNA-alkylating agents (30) each cause the generation of an RPA species that is unable to support DNA replication in vitro. In the case of heat shock, the inhibition of RPA activity is mediated by a stress-dependent association with the nucleolar protein nucleolin (12,47).In an area with potential regulatory significance, R...

under non-stress conditions, the mutant cells were severely deficient in the amount of DNA synthesis occurring during replication stress. These cells also had abnormally high levels of chromatin-bound RPA, indicative of increased amounts of single-stranded DNA (ssDNA) and showed defective recovery from stress. Cells replaced with the mutant RPA2 also generated G1 cells with a broader DNA distribution and high levels of apoptosis following stress, compared with cells expressing wild-type RPA2. Surprisingly, cells expressing the wild-type RPA2 subunit had increased levels of stress-dependent DNA breaks. Our data demonstrate that RPA phosphorylation at the T21 and S33 sites facilitates adaptation of a DNA-replication fork to replication stress. Supplementary material available online at

No abstract