Identification of DNA scission site by gamma ray irradiation at one base pair level

[Introduction]It is known that DNA is damaged by the radiation, and many researchers studied the type of DNA damage, the DNA repair mechanism, and so on. However, identification of scission site and specificity of scission site have not been studied in detail up to now. We, therefore, cleaved DNA by irradiation of ⁶⁰Co gamma ray, and specified the scission sites at one base pair level by using the technique that we developed. We considered the relationship between the scission sites and the type of base as well as DNA secondary structure.[Experimental]Five hundreds ng/ul of pBR322 plasmid DNA(form I) solution was prepared. The solution was irradiated with 60Co gamma ray (0.31 MeV and 1.17 MeV) in the range of 0 to 20 Gy. After irradiation, a small amount of DNA was electrophoresed on agarose gel to confirm whether DNA was cleaved or not. After that, DNA scission site was identified at one base pair level by using the novel method. [Results & Discussion]The experiment of DNA scission by gamma ray irradiation and specification of scission site went twice on the same condition. Both DNA scissions were caused at almost the same sites. As a result, we confirmed reproducibility of DNA scission by gamma ray irradiation. Then the DNA solution was left for several days at room temperature and the scission site was specified. It was confirmed that the scission sites by causing leaving in the room accorded with the scission sites by causing gamma ray irradiation. Further, the secondary structure of DNA containing the scission site was predicted by the software on the market (Genetyx, Inc.). When the scission site was confirmed on the predicted secondary structure, 80% or more of the scission sites were on the loop. It is thought that the base in the loop is unstable. So far it has been thought that there is no positional specificity in DNA scission by radiation. As a result, however, it is suggested that structural instability parts exist in the base sequence of DNA, and that these parts are damaged prior to other parts by gamma ray irradiation. We will introduce that the novel method in detail and discuss that the frequencies of DNA scission can be estimated using this method. [Reference][1]F. Sakamoto et al., J. Biochem. Biophys. Methods, 52, 97-109 (2002).