Induction and repair of DNA base damage studied in X-irradiated CHO cells using the M. luteus extract.

DNA base damage was measured in Chinese hamster ovary cells X-irradiated under aerobic conditions using an extract of the bacterium Micrococcus luteus. The glycosylases and endonucleases present in this extract recognize damaged bases and convert them into strand breaks (termed endonuclease-sensitive sites, enss). Strand breaks were detected by the alkaline unwinding technique. The induction of enss was measured for X-ray doses ranging up to 45 Gy. The relative frequency of all enss related to all radiation induced strand breaks was 1.7 +/- 0.4. Repair of enss was studied for a radiation dose of 45 Gy. The number of enss was found to decrease exponentially with time after irradiation with a half-time of tau enss = 37 +/- 8 min. The repair kinetics that were also measured for all X-ray-induced DNA strand breaks were found to consist of three phases: fast, intermediate and slow. The intermediate phase was fitted under the assumption that this phase results from the formation and repair of secondary single-strand breaks generated by enzymatic incision at the sites of base damage repair. The relative frequency of base damage derived from this fit was 1.8 +/- 0.5 and the half-time of base damage repair was tau in = 32 +/- 6 min. The agreement of this half-time with the half-time obtained when base damage was measured directly using the M. luteus assay gives support to the interpretation that the intermediate phase of the total repair curve represents the kinetics of secondary strand breaks resulting from base damage by enzymatic incision.