Molecular Mechanisms of Pyrimidine Dimer Excision in Saccharomyces cerevisiae : Incision of Ultraviolet-Irradiated Deoxyribonucleic Acid In Vivo

A group of genetically related ultraviolet (UV)-sensitive mutants ofSaccharomyces cerevisiae has been examined in terms of their survival after exposure to UV radiation, their ability to carry out excision repair of pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-UV incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploidRAD + strain (S288C), 11 different mutants representing sixRAD loci (RAD1, RAD2, RAD3, RAD4, RAD14 , andRAD18 ) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation fromM. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. Therad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did therad2-4 mutant. The latter was the only one of three differentrad2 alleles examined which was leaky in this respect. The UV-sensitive strain carrying the mutant allelerad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to theRAD6 rather than theRAD3 epistatic group. All strains having mutant alleles of theRAD1, RAD2, RAD3, RAD4 , andRAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to UV radiation. These experiments suggest that theRAD1, RAD2, RAD3, RAD4 (and probablyRAD14 ) genes are all required for the incision of UV-irradiated DNA during pyrimidine dimer excision in vivo.