Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

Abstract

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...

Mitotic crisis: The unmasking of a novel role for RPA

“…Second, RPA2 phosphorylation in interphase cells stimulates repair of chromosomal DNA damage caused either by camptothecin or bleomycin treatment. 4 Because RPA phosphorylation does not significantly alter the high affinity of RPA for ssDNA, 7,8 a likely scenario is that phosphorylated RPA located at DNA damage sites recruits factors for DNA repair ( Fig. 2A).…”

“…Phosphorylation of the RPA2 subunit proceeds through a favored pathway in interphase cells. 4 Under DNA damage conditions that cause the generation of ssDNA, the resulting persistent RPA-ssDNA entities act to recruit and activate the ATR checkpoint kinase. Co-incident phosphorylation of Ser33 In interphase cells, formation of DSBs causes initial modification of S33 by ATR, stimulating phosphorylation of adjacent S23 and S29 by cyclin/Cdk2.…”

“…8 Even so, test of nonstressed cells in which endogenous RPA2 was 'replaced' with a mutant preventing Cdk phosphorylation (and thus generation of the mitotic RPA form) found only minor effects on the cell cycle profile, as compared to cells replaced with a wt-RPA2 subunit. 4 Testing crisis conditions (i.e., subjecting nocodazole-arrested cells to bleomycin) to unmask the significance of mitotic RPA phosphorylation revealed a novel activity for RPA. Previous studies of cultured mammalian cells observed that mitotic DNA damage causes a delay in anaphase onset, 9 dependent on Brca1 and Chk1 function.…”

Differential response of normal and malignant urothelial cells to CHK1 and ATM inhibitors

“…It has previously been shown that RPA2 has a distinctive pattern of phosphorylation in many tumour cell lines in response to replication and CHK1 inhibitors (Anantha et al, 2007;Liu et al, 2012). There is general agreement that Ser33 is a phosphorylation target for activated ATR (Anantha et al, 2007;Vassin et al, 2009). In contrast, Ser4/8 is mainly phosphorylated through ATM or DNAPK activity (Liu et al, 2012).…”

“…In contrast, Ser4/8 is mainly phosphorylated through ATM or DNAPK activity (Liu et al, 2012). Ser4/8 phosphorylation of RPA is usually found only in the most hyper-phosphorylated forms of RPA2 during DNA replication stress following phosphorylation of Ser33 which is phosphorylated by ATR (Anantha et al, 2007).…”