Involvement of DNA-dependent protein kinase in UV-induced replication arrest

Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G₁/S boundary while repair occurs. It is not known how UV irradiation induces S-phase arrest and yet permits DNA repair; however, UV- induced inhibition of replication is efficiently reversed by the addition of replication protein A (RPA), suggesting a role for RPA in this regulatory event. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), plays a role in UV-induced replication arrest. DNA synthesis of M059K (DNA-PK catalytic subunit-positive (DNA-PKcs⁺)), as measured by [³H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PKcs^-) cells were much less affected. Similar results were obtained with the in vitro replication reactions where immediate replication arrest occurred in DNA-PKcs⁺ cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs^- cells. Reversal of replication arrest was observed at 8 h following UV irradiation in DNA-PKcs⁺ cells but not in DNA-PKcs^- cells. Reversal of UV- induced replication arrest was also observed in vitro by the addition of a DNA-PK inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a positive role for DNA-PK in damage-induced replication arrest. The RPA- containing fraction from UV-irradiated DNA-PKcs⁺ cells poorly supported DNA replication, whereas the replication activity of the RPA-containing fraction from DNA-PKcs^- cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA activity. Together, our results strongly suggest a role for DNA-PK in S-phase (replication) arrest in response to UV irradiation.