SITES OF UV-INDUCED PHOSPHORYLATION OF THE P34 SUBUNIT OF REPLICATIONPROTEIN-A FROM HELA-CELLS

Exposure of mammalian cells to UV radiation alters gene expression and cell cycle progression; some of these responses may ensure survival o r serve as mutation-avoidance mechanisms, lessening the consequences o f UV-induced DNA damage, We showed previously that UV irradiation incr eases phosphorylation of the p34 subunit of human replication protein A (RPA) and that this hyperphosphorylation correlated with loss of act ivity of the DNA replication complex. To characterize further the role of RPA hyperphosphorylation in the cellular response to UV irradiatio n and to determine which protein kinases might be involved, we identif ied by phosphopeptide analysis the sites phosphorylated in the p34 sub unit 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 an d one threonine in the N-terminal 33 residues of RPA-p34 can become ph osphorylated after UV irradiation, Two of these sites (Ser-23 and Ser- 29) ape known to be sites phosphorylated by Cdc2 kinase; two others (T hr-21 and Ser-33) are consensus sites for the DNA-dependent protein ki nase (DNA-PK); the fifth site (Ser-11, -12, or -13) does not correspon d to the (Ser/Thr)-Gln DNA-PK consensus, All five can be phosphorylate d in vitro by incubating purified RPA with purified DNA-PK. Two additi onal sites, probably Ser-4 and Ser-8, are phosphorylated in vivo after UV irradiation and in vitro by purified DNA-PK. The capacity of purif ied DNA-PK to phosphorylate many of these same sites on RPA-p34 in vit ro implicates DNA-PK or a kinase with similar specificity in the UV-in duced hyperphosphorylation of RPA in vivo.