Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV

Abstract XRCC4 and DNA Ligase IV (LIG4) cooperate to join two DNA ends at the final step of DNA double-strand break (DSB) repair through non-homologous end-joining (NHEJ). However, it is not fully understood how these proteins are localized to the nucleus. Here we created XRCC4 K271R mutant, as Lys271 lies within the putative nuclear localization signal (NLS), and XRCC4 K210R mutant, as Lys210 was reported to undergo SUMOylation, implicated in the nuclear localization of XRCC4. Wild-type and mutated XRCC4 with EGFP tag were introduced into HeLa cell, in which endogenous XRCC4 had been knocked down using siRNA directed to 3′-untranslated region, and tested for the nuclear localization function by fluorescence microscopy. XRCC4 K271R was defective in the nuclear localization of itself and LIG4, whereas XRCC4 K210R was competent for the nuclear localization with LIG4. To examine DSB repair function, wild-type and mutated XRCC4 were introduced into XRCC4-deficient M10. M10-XRCC4 K271R , but not M10-XRCC4 K210R , showed significantly reduced surviving fraction after 2 Gy γ-ray irradiation as compared to M10-XRCC4 WT . The number of γ-H2AX foci remaining 2 h after 2 Gy γ-ray irradiation was significantly greater in M10-XRCC4 K271R than in M10-XRCC4 WT , whereas it was only marginally increased in M10-XRCC4 K210R as compared to M10-XRCC4 WT . The present results collectively indicated that Lys271, but not Lys210, of XRCC4 is required for the nuclear localization of XRCC4 and LIG4 and that the nuclear localizing ability is essential for DSB repair function of XRCC4.