Excision repair in u.v. (254 nm) damaged non-dividing human skin fibroblasts: a major biological role for DNA polymerase alpha.
We have used the eukaryotic DNA polymerase alpha inhibitor, aphidicolin, and the polymerase beta inhibitor, dideoxythymidine, to examine the role of these enzymes in excision repair of ultraviolet (u.v., 254 nm) damage induced in non-dividing (arrested) human skin fibroblasts. The effects of these drugs on u.v.-treated cells have been monitored using a simple and reproducible repair synthesis assay in parallel with viability measurements to determine the degree of inhibition of repair of potentially lethal damage. In agreement with previous studies using density gradients, repair synthesis induced by low fluences of u.v. (less than 3 J m-2) is relatively insensitive to inhibition by aphidicolin compared to high fluences where approximately 85 per cent inhibition is observed at the highest (20 micrograms/ml) aphidicolin concentration employed. However, repair of potentially lethal damage is inhibited by at least 90 per cent over the entire fluence range. Although dideoxythymidine led to considerable inhibition of repair synthesis, the result is probably an artifact under these in vivo conditions. The polymerase beta inhibitor was not toxic to u.v.-treated cells nor did it add to the toxicity of aphidicolin when the drugs were used in combination. We conclude that if the beta polymerase is involved in excision repair then its temporary (4 h) inhibition by dideoxythymidine is entirely reversible. In contrast, polymerase alpha appears to be an enzyme essential to the majority of biologically effective excision repair over the entire u.v. fluence range tested.
D016590:Aphidicolin; D004247:DNA / Q000096:biosynthesis; D004257:DNA Polymerase II / Q000502:physiology*; D004260:DNA Repair; D015224:Dideoxynucleosides; D004224:Diterpenes / Q000494:pharmacology; D005347:Fibroblasts / Q000528:radiation effects; D006801:Humans; D012867:Skin / Q000528:radiation effects; D013936:Thymidine / Q000031:analogs & derivatives / Q000494:pharmacology; D014466:Ultraviolet Rays
D015224:Dideoxynucleosides; D004224:Diterpenes; C019336:2',3'-dideoxythymidine; D016590:Aphidicolin; D004247:DNA; D004257:DNA Polymerase II; D013936:Thymidine