Radiation-induced XRCC4 association with chromatin DNA analyzed by biochemical fractionation.
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.
Kamdar
Radhika Pankaj
RP
Research Laboratory for Nuclear Reactors and Department of Nuclear Engineering, Tokyo Institute of Technology, Tokyo, Japan.
Matsumoto
Yoshihisa
Y
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
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