Abstract
Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. After incubation for 20 h, DNA was recovered from the oocytes and analyzed simultaneously for repair and for intermolecular homologous recombination by electrophoresis and bacterial transformation. Oocyte-mediated repair of DNA strand breaks was observed with both methods. Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we also demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays. X-irradiation of a circular DNA stimulated its potential to recombine with a homologous linear partner. Recombination products were detected directly by Southern blot hybridization and as bacterial transformant clones expressing two antibiotic resistance markers originally carried separately on the two substrates. The increase in recombination was dependent on X-ray dose. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. In summary, oocytes have considerable capacity to repair X-ray-induced damage, and some X-ray lesions stimulate homologous recombination in these cells.
Highlights
Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes
Using a repair-deficient mutant Escherichia coli strain and its repair-proficient parent as hosts for the transformation assay, we demonstrated that oocytes repaired oxidative-type DNA base damage induced by X-rays
There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination
Summary
Plasmid DNA substrates were X-irradiated and injected into the nuclei of Xenopus laevis oocytes. There is some suggestion that lesions other than double-strand breaks contribute to the stimulation of oocyte-mediated homologous recombination. Double-strand breaks represent at most a few percent of the total damage induced by X rays; single-strand breaks and oxidative-type base lesions (such as thymine glycols, urea residues, etc.) make up 85 to 90% of the remainder [14, 35]. The contribution of the latter lesions to radiation-induced recombination has not been determined. Since the oocyte contains large stores of the proteins and enzymes involved in DNA metabolism [8, 41], it is a promising system for the future development of cell extracts
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