A Novel technique using DNA denaturation to detect prompt and enzymatically induced single strand breaks produced in a clustered damage site

Yokoya, Akinari; Shikazono, Naoya; Urushibara, Ayumi*; Fujii, Kentaro

In order to observe multiply single strand breaks induced by radiation in a clustered DNA damage site, we have developed a novel technique using DNA denaturation. The multiple SSBs which arise in both strands of plasmid DNA, but do not induce a DSB, are measured as decrease of the intact single strand DNA (SS-DNA) using agarose gel electrophoresis. To avoid induction of heat labile damage, we have determined lower denaturation temperature (37 C) using formamide (50% v/v). We have tested the validity of the new assay method by applying to the X-irradiated diluted DNA solution in which SSBs are mainly induced by OH radicals. The dose-response curve of the remaining SS-DNA fraction shows precisely a half slope of that for the closed-circular DNA as theoretically expected. This result indicates that the assay properly works without any artificial damages by the denaturation treatment. The obtained data for several radiations and experimental conditions will be presented.