A sensitive, enzymatic assay for the detection of closely opposed cyclobutyl pyrimidine dimers induced in human diploid fibroblasts.
A sensitive, enzymatic assay has been developed for the detection of closely opposed cyclobutyl pyrimidine dimers induced in UV-irradiated human diploid fibroblasts. In this assay closely opposed dimers are detected as bifilar enzyme-sensitive sites. Single-strand incisions are made at the positions of individual pyrimidine dimers through the action of M. luteus pyrimidine dimer-DNA glycosylase. Incisions at closely opposed dimers, effectively expressed as double-strand breaks, are quantified from the resulting reduction in DNA double-strand molecular weight as determined by velocity sedimentation through neutral sucrose density gradients. The stability of the bacteriophage lambda cos site under our reaction conditions indicates that opposed incisions must be relatively close to be expressed as a double-strand break. The dose response for the induction of bifilar enzyme-sensitive sites in mammalian cells was found to be complex but can be approximated by a function that increases as the 1.2-1.4 power of UV dose. The frequency of bifilar enzyme-sensitive sites observed decreased during postirradiation incubation of excision-repair-proficient human diploid fibroblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m2 (254 nm). By comparison, over 80% of the bifilar enzyme-sensitive sites induced in fibroblasts assigned to xeroderma pigmentosum complementation group A remained detectable 24 h after irradiation. The implications of these results for models addressing the induction and repair of closely opposed pyrimidine dimers are discussed.
D002478:Cells, Cultured; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000528:radiation effects; D006801:Humans; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D011740:Pyrimidine Dimers / Q000032:analysis*; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics
D011740:Pyrimidine Dimers; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C036401:deoxyribopyrimidine endonucleosidase