title: Detection of DNA double-strand breaks by pulsed-field gel electrophoresis.
abstract: A DNA double-strand break (DSB) is one of the most cytotoxic DNA lesions because unrepaired DSBs cause chromosomal aberrations and cell death. Although many physiological DSBs occur at DNA replication sites, the molecular mechanisms underlying this remain poorly understood. There was therefore a need to develop a highly specific method to detect DSB fragments containing DNA replication sites. Here we investigated whether pulsed-field gel electrophoresis (PFGE) combined with visualization of DNA replication sites by immunoblotting using halogenized deoxyuridines, such as BrdU and IdU, was sufficient for this detection. Our methodology enabled us to reproduce previously reported data. In addition, this methodology was also applied to the detection of bacterial infection-induced DSBs on human chromosomal DNA. Based on our findings, we propose that this strategy combining PFGE with immunoblot analysis will be applicable to studies analyzing the mechanistic details of DNA repair, the DNA damage response and the activity of DNA-damaging agents.
authors.affiliation: Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, Japan.|Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Kawashima
authors.affiliation: Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Nahomi
authors.initials: N
authors.lastname: Yamaguchi
authors.affiliation: Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Teshima
authors.affiliation: Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narahara
authors.affiliation: Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamaoka
authors.affiliation: Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Anai
authors.affiliation: Department of Obstetrics and Gynecology, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Nishida
authors.affiliation: Department of Environmental and Preventive Medicine, Faculty of Medicine, Oita University, Yufu, Japan.|Clinical Engineering Research Center, Faculty of Medicine, Oita University, Yufu, Japan.
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Hanada
mesh_terms: D001973:Bromodeoxyuridine / Q000494:pharmacology; D002875:Chromosomes / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D016521:Electrophoresis, Gel, Pulsed-Field; D006801:Humans
chemical_list: D004247:DNA; D001973:Bromodeoxyuridine