We show that DNA replication activity in extracts of human HeLa cells decreases following UV irradiation. Alterations in replication activity in vitro parallel the UV‐induced block in cell cycle progression of these cells in culture. UV irradiation also induces specific changes in the pattern of phosphorylation of the 34 kDa subunit of a DNA replication protein, human single‐stranded DNA‐binding protein (hSSB). The appearance of a hyperphosphorylated form of hSSB correlates with reduced in vitro DNA replication activity in extracts of UV‐irradiated cells. Replication activity can be restored to these extracts in vitro by addition of purified hSSB. These results suggest that UV‐induced DNA synthesis arrest may be mediated in part through phosphorylation‐related alterations in the activity of hSSB, an essential component of the DNA replication apparatus.

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...

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

We have used an SV40-based shuttle vector, pZ189, to investigate the capacity of HeLa cell extracts to reproduce the in vivo process of mutation fixation. We showed previously that when UV-irradiated pZ189 is replicated in these extracts, bypass of UV photoproducts occurs, resulting in base substitution mutations in the supF gene of the vector. Here we report the DNA sequence characterization of a collection of 60 of these UV-induced mutants. Most of the mutations observed are single or tandem double base substitutions at dipyrimidine sites; of these, approximately 90% are G:C-->A:T transitions. Mutations are observed predominantly at a few sites, in particular at positions 155 and 156 in the supF sequence. No dramatic differences in the mutation spectrum were observed when the orientation of the supF gene was reversed with respect to the SV40 origin of replication, suggesting that mutation fixation occurs similarly on both the leading and the lagging strands for DNA replication. Generally, the mutational hot spots observed in vitro are at the same sites as those observed when UV-irradiated pZ189 was passaged in human or monkey cells in culture. Thus, it appears that the replication and mutagenesis of UV-damaged templates in HeLa cell extracts accurately reflects these processes in the intact cell.

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