Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay.
A sensitive and quantitative procedure for the detection of pyrimidine dimers in yesast nuclear DNA is described. The assay employs dimer-specific, endonuclease activities from Micrococcus luteus together with DNA sedimentation through calibrated, alkaline sucrose gradients to detect endonuclease-induced, single-strand breaks. Breaks were induced in a dose-dependent manner from 0 to 80 J m-2 at 254 nm and in numbers equivalent to the numbers of dimers induced by similar doses (Unrau et al., Biochim. Biophys. Acta, 312 (1973) 626--632). This procedure also allows the use of [6-3H] uridine to label cellular nucleic acids, but dose not require extensive DNA purification to eliminate concomitantly labeled RNA. Endonuclease-sensitive sites in the wild-type, haploid strain S288C, after irradiation with 5 J m-2 (254 nm), were removed in less than 5 min when cells were incubated in buffer (pH 7.0) at 28 degrees C. After irradiation with doses from 30 to 100 Jm-2 site removal in S288C required longer postirradiation incubations and was about 90% complete. In a radiation-sensitive strain carrying the mutant allele rad4-3 the number of endonuclease-sensitive sites remained constant for 6 h after irradiation with 5 Jm-2. The retention of sites in this strain indicates that it is defective in the excision of pyrimidine dimers.
D002499:Centrifugation, Density Gradient; D004247:DNA / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D004720:Endonucleases / Q000378:metabolism; D008722:Methods; D008837:Micrococcus / Q000201:enzymology; D011740:Pyrimidine Dimers / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000528:radiation effects; D014466:Ultraviolet Rays
D011740:Pyrimidine Dimers; D004247:DNA; D004720:Endonucleases