Replication-mediated DNA damage by camptothecin induces phosphorylation ofRPA by DNA-dependent protein kinase and dissociates RPA : DNA-PK complexes

Replication protein A (RPA) is a DNA single-strand binding protein essentia l for DNA replication, recombination and repair. In human cells treated wit h the topoisomerase inhibitors camptothecin or etoposide (VP-16), we find t hat RPA2, the middle-sized subunit of RPA, becomes rapidly phosphorylated, This response appears to be due to DNA-dependent protein kinase (DNA-PK) an d to be independent of p53 or the ataxia telangiectasia mutated (ATM) prote in. RPA2 phosphorylation in response to camptothecin required ongoing DNA r eplication. Camptothecin itself partially inhibited DNA synthesis, and this inhibition followed the same kinetics as DNA-PK activation and RPA2 phosph orylation, DNA-PK activation and RPA2 phosphorylation were prevented by the cell-cycle checkpoint abrogator 7-hydroxystaurosporine (UCN-01), which mar kedly potentiates camptothecin cytotoxicity. The DNA-PK catalytic subunit ( DNA-PKcs) was found to bind RPA which was replaced by the Ku autoantigen up on camptothecin treatment. DNA-PKcs interacted directly with RPA1 in vitro. We propose that the encounter of a replication fork with a topoisomerase-D NA cleavage complex could lead to a juxtaposition of replication fork-assoc iated RPA and DNA double-strand end-associated DNA-PK, leading to RPA2 phos phorylation which may signal the presence of DNA damage to an S-phase check point mechanism.