Abstract
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 λ 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 fibriblasts with less than 20% still detectable at 24 h after irradiation with 5 J/m 2 (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.
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