Measurement of multiple SSB site in plasmid DNA by high-LET irradiation

We reported that the yields base lesions which are recognized by base excision repair enzymes such as Nth and Fpg (glycosylases) strikingly decreased with increasing level of LET value of radiation (this conference in 2006). Multiply induced DNA damages composed of SSBs and base lesions (clustered damage) by high LET radiation are thought to compromise the enzymatic activities. One of the goals of our study is to clarify the structure of clustered DNA damage. Many studies using plasmid DNA, however, does have a technical limitation. The number of SSBs in a plasmid DNA molecule would be underestimated since these SSBs including enzymatically induced ones will not cause additional conformational changes if they are on the same strand or on the opposite strand but separated each other sufficiently (>6 bp) so as not to induce a DSB. In order to observe these secondary invisible SSBs, we have developed a novel technique using DNA denaturation by which irradiated DNA is analyzed as single strand DNA (SS-DNA)(this conference in 2007). Fully hydrate pUC18 plasmid DNA was exposed to He ion beam from (82 keV/m) TIARA (JAEA-TAKASAKI). After denaturation treatment at 37 C using formamide (50% v/v) for 5 min, remaining fraction of intact SS-DNA was analyzed by gel electrophoresis (1% agarose) and quantified by staining with EtBr. Obtained result shows that multiple SSBs in both DNA strand are hardly induced by high LET irradiation. This indicates that clustered DNA damage would be mainly composed of one SSB and one (or multiple) base lesion(s).