Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group

Rejoining of ionizing radiation (IR) induced DNA DSBs usually follows bipha sic kinetics with a fast (t(50): 5-30 min) component attributed to DNA-PK-d ependent non-homologous endjoining (NHEJ) and a slow (t(50): 1-20 h), as of yet uncharacterized, component. To examine whether homologous recombinatio n (HR) contributes to DNA DSB rejoining, a systematic genetic study was und ertaken using the hyper-recombinogenic DT40 chicken cell line and a series of mutants defective in HR, We show that DT40 cells rejoin IR-induced DNA D SBs with half times of 13 min and 4.5 h and contributions by the fast (78%) and the slow (22%) components similar to those of other vertebrate cells w ith 1000-fold lower levels of HR, We also show that deletion of RAD51B, RAD 52 and RAD54 leaves unchanged the rejoining half times and the contribution of the slow component, as does also a conditional knock out mutant of RAD5 1, A significant reduction (to 37%) in the contribution of the fast compone nt is observed in Ku70 / DT40 cells, but the slow component, operating with a half time of 18.4 h, is still able to rejoin the majority (63%) of DSBs, ri double mutant Ku70(-/-)/RAD54 / shows similar half times to Ku70(-/-) c ells. Thus, variations in HR by several orders of magnitude leave unchanged the kinetics of rejoining of DNA DSBs, and fail to modify the contribution of the slow component in a way compatible with a dependence on HR, We prop ose that, in contrast to yeast, cells of vertebrates are 'hard-wired' in th e utilization of NHEJ as the main pathway for rejoining of IR-induced DNA D SBs and speculate that the contribution of homologous recombination repair (HRR) is at a stage after the initial rejoining.