title: A novel and simple micro-irradiation technique for creating localized DNA double-strand breaks.
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.
authors.affiliation: Atomic Bomb Disease Institute, Course of Life Sciences and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523, Japan. kzsuzuki@net.nagasaki-u.ac.jp
authors.forename: Keiji
authors.initials: K
authors.lastname: Suzuki
authors.forename: Motohiro
authors.initials: M
authors.lastname: Yamauchi
authors.forename: Yasuyoshi
authors.initials: Y
authors.lastname: Oka
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Suzuki
authors.forename: Shunichi
authors.initials: S
authors.lastname: Yamashita
mesh_terms: D000818:Animals; D001973:Bromodeoxyuridine; D002478:Cells, Cultured; D006224:Cricetinae; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004261:DNA Replication; D006801:Humans; D008567:Membranes, Artificial; D008856:Microscopy, Fluorescence; D014466:Ultraviolet Rays
chemical_list: D008567:Membranes, Artificial; D001973:Bromodeoxyuridine
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