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 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 (DNA-PKcs(+))), as measured by [(3)H]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.
Park
J S
JS
Department of Biochemistry and Molecular Biology, the Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
Park
S J
SJ
Peng
X
X
Wang
M
M
Yu
M A
MA
Lee
S H
SH
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D051379:Mice; D016513:Mice, SCID; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014466:Ultraviolet Rays; D000077191:Wortmannin
D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin