title: Radiation-induced XRCC4 association with chromatin DNA analyzed by biochemical fractionation.
abstract: XRCC4, in association with DNA ligase IV, is thought to play a critical role in the ligation of two DNA ends in DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ) pathway. In the present study, we captured radiation-induced chromatin-recruitment of XRCC4 by biochemical fractionation using detergent Nonidet P-40. A subpopulation of XRCC4 changed into a form that is resistant to the extraction with 0.5% Nonidet P-40-containing buffer after irradiation. This form of XRCC4 was liberated by micrococcal nuclease treatment, indicating that it had been tethered to chromatin DNA. This chromatin-recruitment of XRCC4 could be seen immediately (< 0.1 hr) after irradiation and remained up to 4 hr after 20 Gy irradiation. It was seen even after irradiation of small doses, i.e., 2 Gy, but the residence of XRCC4 on chromatin was very transient after 2 Gy irradiation, returning to near normal level in 0.2-0.5 hr after irradiation. The chromatin-bound XRCC4 represented only approximately 1% of total XRCC4 molecules even after 20 Gy irradiation and the quantitative analysis using purified protein as the reference suggested that only a few XRCC4-DNA ligase IV complexes were recruited to each DNA end. We further show that the chromatin-recruitment of XRCC4 was not attenuated by wortmannin, an inhibitor of DNA-PK, or siRNA-mediated knockdown of the DNA-PK catalytic subunit (DNA-PKcs), indicating that this process does not require DNA-PKcs. These results would provide us with useful experimental tools and important insights to understand the DNA repair process through NHEJ pathway.
authors.affiliation: Research Laboratory for Nuclear Reactors and Department of Nuclear Engineering, Tokyo Institute of Technology, Tokyo, Japan.
authors.forename: Radhika Pankaj
authors.initials: RP
authors.lastname: Kamdar
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Matsumoto
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002843:Chromatin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics* / Q000378:metabolism; D051747:DNA-Activated Protein Kinase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019583:Dose Fractionation, Radiation; D004307:Dose-Response Relationship, Radiation; D051379:Mice; D008836:Micrococcal Nuclease / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D000077191:Wortmannin; D014965:X-Rays
chemical_list: D000730:Androstadienes; D002843:Chromatin; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C101467:XRCC4 protein, human; D004247:DNA; D051747:DNA-Activated Protein Kinase; D008836:Micrococcal Nuclease; D000077191:Wortmannin