The study of repair of plasmid DNA treated with 8-methoxypsoralen plus near UV-light (λ=365 NM) in cells of wild type and rad2 mutant of Saccharomyces cerevisial

The method of repeated irradiation has been used to study excision of 8-methoxypsoralen 8-MPP monoadducts from plasmic and chromosomal DNA in cells of wild type and rad2 mutant of Saccharomyces cerevisiae. The measurements of kinetics of monoadduct removal from chromosomal DNA in intact and competent yeast cells showed that monoadducts were excited in both types of cells with normal repair, but this process was blocked in intact and competent cells of the rad2 mutant. The survival of pYF91 plasmic treated in vitro with 8-MOP plus near UV-light has been studied in the cells of the wild type and in incision-defective rad2 mutant by the measurement of cell transformation frequency. Episomic pYF91 plasmid used in these experiments contained the yeast nuclear LEU2 gene, a portion of 2 mkm DNA and DNA of bacterial plasmid pBR322 with resistance to ampicillin. The pYF91 plasmid was treated with 8-MOP plus near UV-light in vitro, then unbound 8-MOP was removed by dialysis. This DNA was used for transformation. The transformed yeast cells were irradiated repeatedly. The quantitative alteration of the yield of transformants, depending on the time of keeping these yeast cells in complete liquid medium at 30 deg C prior to repeated irradiation, allowed to measure the kinetics of monoadduct excision from plasmid DNA. It was shown that monoadducts were removed equally effectively from plasmid DNA introduced into cells of the wild type and rad2 mutant. Possibly, the repair system of both these strains provides excision of monoadducts from plasmic DNA, but this processes blocked in the rad2 mutant, relatively to monoadduct excision from chromosomal DNA