Sites of UV-induced Phosphorylation of the p34 Subunit of Replication Protein A from HeLa Cells

Abstract

Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival or serve as mutation-avoidance mechanisms, lessening the consequences of UV-induced DNA damage. We showed previously that UV irradiation increases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of activity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiation and to determine which protein kinases might be involved, we identified by phosphopeptide analysis the sites phosphorylated in the p34 subunit of RPA (RPA-p34) from HeLa cells before and after exposure to 30 J/m 2 UV light. In unirradiated HeLa cells, RPA-p34 is phosphorylated primarily at Ser-23 and Ser-29. At least four of the eight serines and one threonine in the N-terminal 33 residues of RPA-p34 can become phosphorylated after UV irradiation. Two of these sites (Ser-23 and Ser-29) are known to be sites phosphorylated by Cdc2 kinase; two others (Thr-21 and Ser-33) are consensus sites for the DNA-dependent protein kinase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspond to the (Ser/Thr)-Gln DNA-PK consensus. All five can be phosphorylated in vitro by incubating purified RPA with purified DNA-PK. Two additional sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purified DNA-PK to phosphorylate many of these same sites on RPA-p34 in vitro implicates DNA-PK or a kinase with similar specificity in the UV-induced hyperphosphorylation of RPA in vivo.Replication protein A (RPA 1 ; also known as human SSB) is a trimeric single-stranded DNA-binding protein necessary for DNA replication (1-3), recombination (4), and repair (5, 6).RPA binds to DNA through its 70-kDa subunit (7). In vitro, the DNA binding activity of RPA is essential for DNA unwinding at the origin of replication. The p70 subunit alone, or SSB proteins from other species (8 -12), also can cause unwinding, but specific protein-protein interactions between RPA and polymerase ␣-primase and SV40 large tumor antigen are necessary for assembly of the initiation complex at the SV40 origin of replication and for DNA replication. Neither RPA-p70 alone nor heterologous SSBs substitute for RPA in these interactions (13). Thus, the RPA-p34 and RPA-p13 subunits are primarily responsible for the specificity of protein-protein interactions in DNA replication and possibly also in DNA repair. The RPA protein was shown to interact with XPA (XP group A) (14 -16), XPF-ERCC1 (XPF-excision repair cross-complementing rodent repair deficiency 1 (XP group F)) (17) and XPG (XP group G) (15, 17) proteins in excision repair. RPA-p34 is phosphorylated in vitro by DNA-PK, a DNA-activated protein kinase that participates in double-strand break repair (18 -24). RPA also interacts with several transcription factors, including VP16, Gal4, and...

ATM: A mediator of multiple responses to genotoxic stress

“…The 34 kDa RPA subunit, RPA2, is phosphorylated by Cdc2 on Ser-23 and Ser-29 during S and G2 phases of the cell cycle. In response to DNA damage, RPA2 is further phosphorylated on additional sites within the Nterminus (Zernik-Kobak et al, 1997). Although this N-terminal domain is not absolutely required for ssDNA binding or DNA replication, its phosphorylation a ects the structure of the RPA complex, and probably regulates RPA function by modulating protein ± protein interactions (Braun et al, submitted).…”

DNA Damage Induced Hyperphosphorylation of Replication Protein A. 1. Identification of Novel Sites of Phosphorylation in Response to DNA Damage

“…These two DNA binding domains are termed DBD A and B and the structure of which has been solved by X-ray crystallography in the presence and absence of DNA (11,12). Evidence also suggests that the central domain of RPA-p34 and the C-terminal domain of RPA-p70, DBDs D and C, respectively, may contribute to DNA binding affinity (13).It is well documented that RPA is phosphorylated in vivo with the N-terminus of RPA-p34 being the most well characterized region of RPA modification (14,15). Normally cycling HeLa cells show two RPA-p34 bands separated by single dimensional SDS-PAGE.…”

Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

“…Ser 23 and Ser 29 are constitutively modified during mitosis by cyclin B-Cdk1 (18,19) and have been suggested to be partially modified beginning at the G 1 /S boundary by the cyclin A-Cdk2 complex (18,20,21). These two residues may also undergo heightened phosphorylation in response to UV irradiation (22). The Thr 21 and Ser 33 residues are consensus sites for phosphatidylinositol 3-kinase-like kinase (PIKK) family members (ATM, ATR, and DNA-PK) that signal the presence of DNA damage and replication stress.…”

“…The Thr 21 and Ser 33 residues are consensus sites for phosphatidylinositol 3-kinase-like kinase (PIKK) family members (ATM, ATR, and DNA-PK) that signal the presence of DNA damage and replication stress. Under DNA damage conditions, the phosphorylation of Thr 21 is apparently catalyzed by ATM, DNA-PK, and probably ATR (22,23 . Others have also shown the involvement of DNA-PK in supporting RPA2 hyperphosphorylation (25,26).…”