title: Creating localized DNA double-strand breaks with microirradiation.
abstract: We describe a protocol for creating localized DNA double-strand breaks (DSBs) with minimal requirements that can be applied in cell biology and molecular biology. This protocol is based on the combination of 5-bromo-2'-deoxyuridine (BrdU) labeling and ultraviolet C (UVC) irradiation through porous membranes. Cells are labeled with 10 μM BrdU for 48-72 h, washed with Ca(2+)- and Mg(2+)-free PBS(-), covered by polycarbonate membranes with micropores and exposed to UVC light. With this protocol, localized DSBs are created within subnuclear areas, irrespective of the cell cycle phase. Recruitment of proteins involved in DNA repair, DNA damage response, chromatin remodeling and histone modifications can be visualized without any specialized equipment. The quality is the same as that obtained by laser microirradiation or by any other focal irradiation. DSBs become visible within 30 min of UVC irradiation.
authors.affiliation: Atomic Bomb Disease Institute, Course of Life Sciences and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan. kzsuzuki@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: D001973:Bromodeoxyuridine / Q000737:chemistry; D053903:DNA Breaks, Double-Stranded; D008851:Micropore Filters; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D014466:Ultraviolet Rays
chemical_list: D001973:Bromodeoxyuridine