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      • Involvement of DNA-dependent Protein Kinase in UV-induced Replication Arrest

        Park, Jang-Su,Park, Su-Jung,Peng, Xiaodong,Wang, Mu,Yu, Myeing-Ae,Lee, Suk-Hee 부산대학교 유전공학연구소 1999 분자생물학 연구보 Vol.15 No.-

        Cells exposed to UV irradiation are predominantly arrested at S-phase as well as at the G_1/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 (DNS-PKcs^+)), as measured by [^3H]thymidine incorporation, was significantly arrested by 4 h following UV irradiation, whereas M059J (DNA-PK^-) 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 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.

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