Abstract
An X-ray microbeam irradiation system using synchrotron radiation has been developed at the Photon Factory, KEK, Japan. By using this system, localization of the proteins related DNA repair in cells irradiated with subcellular-sized X-ray beam was investigated. GFP (green fluorescent protein) is useful to visualize the localization of the protein in "living" cells. We constructed CHO cells carrying GFP-tagged Rad51, which is involved in the homologous recombination, one of the main pathways for DNA double strand break repair. After microbeam irradiation, GFP-Rad51 foci were observed with a laser confocal microscope.
Within 1 hour after irradiation of X-ray microbeam, the dose of which was equivalent to 2-5 Gy, fluorescent foci of GFP-Rad51 could be observed at the microbeam-irradiated site in the significant proportion of the irradiated cells. After microscopic observation at 1hr after irradiation, cells were fixed and stained by propidium iodide (PI) to quantitate DNA contents for cell cycle determination. Vast majority of the cells without foci at 1 hour after irradiation was in G1 phase.
Time course of GFP-Rad51 foci formation and disappearance was observed. At 40min after irradiation, GFP-Rad51 foci started appearing in S/G2/M cells. The numbers of the foci increased (~2 hrs) and then decreased (~8 hrs). At 12 hrs after irradiation or later, the foci in G1 cells started appearing.
