UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key prote in in the cellular processes of nucleotide excision repair, DNA recombinati on, and DNA double-strand break repair is the single-stranded DNA binding p rotein, RPA. We showed previously that the p34 subunit of RPA becomes hyper phosphorylated 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 expr ession of ATM, the product of the gene mutated in the human genetic disorde r 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 co mplex in vitro at many of the same sites that are phosphorylated in vivo af ter 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 tha t this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photopro ducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a partici pant in the recombinational postreplication repair of these replication pro ducts. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a co nsequence of UV radiation.