Replication protein A (RPA, also known as human
single-stranded DNA-binding protein) is a trimeric, multifunctional
protein complex involved in DNA replication, DNA repair, and recombination. Phosphorylation of the RPA2 subunit is observed after exposure of cells to ionizing radiation (IR) and other
DNA-damaging agents, which implicates the modified
protein in the regulation of DNA replication after DNA damage or in DNA repair. Although
ataxia telangiectasia-mutated (ATM) and
DNA-dependent protein kinase (
DNA-PK) phosphorylate RPA2 in vitro, their role in vivo remains uncertain, and contradictory results have been reported. Here we show that RPA2 phosphorylation is delayed in cells deficient in one of these
kinases and completely abolished in wild-type, ATM, or
DNA-PK-deficient cells
after treatment with
wortmannin at a concentration-inhibiting ATM and
DNA-PK.
Caffeine, an inhibitor of ATM and ATM-Rad3 related (ATR) but not
DNA-PK, generates an
ataxia-telangiectasia-like response in wild-type cells, prevents completely RPA2 phosphorylation in
DNA-
PKcs deficient cells, but has no effect on
ataxia-telangiectasia cells. These observations rule out ATR and implicate both ATM and
DNA-PK in RPA2 phosphorylation after exposure to IR.
UCN-01, an inhibitor of
protein kinase C, Chk1, and
cyclin-dependent kinases, has no effect on IR-induced RPA2 phosphorylation. Because
UCN-01 abrogates checkpoint responses, this observation dissociates RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA has a higher affinity for nuclear structures than unphosphorylated RPA suggesting functional alterations in the
protein. In an in vitro assay for DNA replication,
DNA-PK is the sole
kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation kinetics are distinct from those of the S phase checkpoint, we propose that
DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage to redirect the functions of the
protein from DNA replication to DNA repair.