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 ~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.