Host: The Japan Radiation Research Society
One of the goals of our study is to clarify the nature of clustered damage induced by densely ionizing radiation. Many studies on DNA damage to date have focused on yields of single-(SSB) and double-strand breaks (DSB) in closed-circular plasmid DNA. Oxidative base lesions and cluster damage composed of them have also been analyzed using base excision repair enzymes, which convert a base lesion to a detectable strand break. These studies, however, does have some limitations. For instance, previous simulation studies have shown that the proportion of SSB which are complex, i.e. one or more lesions close to SSB terminus, is significant. The complex SSBs as well as enzymatically induced SSBs, would be underestimated since these SSBs will not cause additional conformational changes if it is on the same strand as the prompt SSB or on the opposite strand but separated sufficiently (>6 bp) from the prompt SSB so as not to induce a DSB. In order to observe these secondary invisible SSBs, we developed a novel technique using DNA denaturation by which irradiated DNA is analyzed as single strand DNA. The prompt or enzymatically revealed additional SSBs which arise in both strands of pUC18 plasmid DNA, but do not induce a DSB, are measured as degradation of single strand (SS-DNA) using gel electrophoresis. To avoid induction of heat labile strand breaks by high temperature generally used for a denaturation treatment, we have determined much lower denaturation temperature (37 degrees centigrade) using formamide (50% v/v). Obtained preliminary results will be presented.
