Abstract
An ataxia-telangiectasia mutated (ATM)-dependent DNA damage signal is amplified through the interaction of various factors, which are recruited to the chromatin regions with DNA double-strand breaks. Spatial and temporal regulation of such factors is analysed by fluorescence microscopy in combination with laser micro-irradiation. Here we describe a novel and simple technique for micro-irradiation that does not require a laser source. Cells were labelled with BrdU for 48-72 h, covered with porous polycarbonate membranes, and exposed to UVC. All BrdU-labelled cells showed localized foci of phosphorylated ATM, phosphorylated histone H2AX, MDC1 and 53BP1 upon irradiation, showing that these foci were induced irrespective of the cell-cycle phase. They were also detectable in nucleotide excision repair-defective XPA cells labelled with BrdU, indicating that the foci did not reflect an excision repair-related process. Furthermore, an ATM-specific inhibitor significantly attenuated the foci formation, and disappearance of the foci was significantly abrogated in non-homologous end-joining-defective cells. Thus, it can be concluded that micro-irradiation generated DNA double-strand breaks in BrdU-sensitized cells. The present technique should accelerate research in the fields of DNA damage response, DNA repair and DNA recombination, as it provides more chances to perform micro-irradiation experiments without any specific equipment.