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

Cells exposed to UV irradiation are predominantly arrested at S-phase as we b as at the G(1)/S boundary while repair occurs. It is not known how UV irr adiation induces S-phase arrest and yet permits DNA repair; however, UV-ind uced inhibition of replication is efficiently reversed by the addition of r eplication protein A (RPA), suggesting a role for RPA in this regulatory ev ent. Here, we show evidence that DNA-dependent protein kinase (DNA-PK), pla ys a role in W-induced replication arrest, DNA synthesis of M059K (DNA-PK c atalytic subunit-positive (DNA-PKcs(+))), as measured by [H-3]thymidine inc orporation, was significantly arrested by 4 h following UV irradiation, whe reas M059J (DNA-PKcs(-)) cells were much less affected. Similar results wer e obtained with the in vitro replication reactions where immediate replicat ion arrest occurred in DNA-PKcs(+) cells following UV irradiation, and only a gradual decrease in replication activity was observed in DNA-PKcs(-) cel ls. Reversal of replication arrest was observed at 8 h following UV irradia tion in DNA-PKcs(+) cells but not in DNA-PKcs(-) cells. Reversal of W-induc ed replication arrest was also observed in vitro by the addition of a DNA-P K inhibitor, wortmannin, or by immunodepletion of DNA-PKcs, supporting a po sitive role for DNA-PK in damage-induced replication arrest. The RPA-contai ning fraction from UV-irradiated DNA-PKcs(+) cells poorly supported DNA rep lication, whereas the replication activity of the RPA-containing fraction f rom DNA-PKcs(-) cells was not affected by UV, suggesting that DNA-PKcs may be involved in UV-induced replication arrest through modulation of RPA acti vity. Together, our results strongly suggest a role for DNA-PK in S-phase ( replication) arrest in response to UV irradiation.