Live cell observation of DNA repair using EGFP plasmid exposed to X-rays in various conditions

Clustered DNA damage, which is defined as two or more lesions (base lesions, single strand breaks or apurinic/apyrimidinic(AP) sites) localized within one or two helical turn(s), is mainly induced by the direct deposition of radiation energy to DNA . Repair susceptibility of clustered DNA damage has not been clarified yet. To elucidate its repair susceptibility, in the present study, fully hydrated DNA films containing 35 water molecules per nucleotide, as well as DNA solutions were used as samples. In the hydrated DNA films, the major process of radiation action is direct ionization or impact of secondary electrons to DNA, because diffusible water radicals are hardly generated. Thus, clustered DNA damage yield in hydrated DNA films is much higher than that produced in DNA solution. The EGFP-expressing plasmids were exposed to X-rays with a 1/e dose that causes loss of the closed circular form resulting 37% of residual intact plasmids, and then transfected into non-irradiated human cells (MCF-7). Those cells were observed for 48h with a fluorescence microscope. DNA repair efficiency was obtained as the number of fluorescence expression cells. The efficiency for the irradiated DNA film was approximately 20% lower than that for the solution sample. These results indicate that DNA damage induced in the hydrated DNA films would be less repaired because of complexity of the damage, namely clustered damage, produced by direct effect.第3回QST国際シンポジウム「Quantum Life Science