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.