Use of Damaged Plasmid to Study DNA Repair in X-ray Sensitive (Xrs) Strains of Chinese Hamster Ovary (CHO) Cells

Summary

The effect of γ-irradiation of pSV2gpt DNA on its transfection frequency has been analysed using CHO xrs mutants. Xrs mutants are sensitive to ionizing radiation and show a defect in double-strand break (dsb) rejoining. At low doses a sharp decrease in relative transfection frequency, i.e. transfection frequency of irradiated plasmid relative to untreated plasmid, was observed in the xrs mutants compared with the parent line K1. Electrophoresis of the irradiated plasmid DNA showed that the decrease in transfection frequency in the xrs mutants correlated with the change of supercoiled molecules into open-circular forms. One explanation for these results is that the xrs gene could play a part in the integration or repair of open-circular molecules produced by γ-radiation. In the parent line CHO-K1, open-circular and supercoiled molecules have the same transfection frequency. The effect of linearization of pSV2gpt DNA by restriction enzymes on transfection frequency in xrs and wild-type strains has also been examined. In contrast to the above results we have not detected a difference in the relative transfection frequency between xrs and wild-type strains. The results suggest that restricted plasmid DNA is subject to extensive nucleolytic degradation, and this occurs to equal extents in wild type and mutant strains.