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-irradiat ed under aerobic conditions using an extract of the bacterium Micrococ cus luteus. The glycosylases and endonucleases present in this extract recognize damaged bases and convert them into strand breaks (termed e ndonuclease-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 re lated 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 ha lf-time of tau(enss)=37+/-8 min. The repair kinetics that were also me asured for all X-ray-induced DNA strand breaks were found to consist o f three phases: fast, intermediate and slow. The intermediate phase wa s fitted under the assumption that this phase results from the formati on and repair of secondary single-strand breaks generated by enzymatic incision at the sites of base damage repair. The relative frequency o f 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 halftime obtained when base damage was measured directl y using the M. luteus assay gives support to the interpretation that t he intermediate phase of the total repair curve represents the kinetic s of secondary strand breaks resulting from base damage by enzymatic i ncision.