Fukuchi Mikoto, Wanotayan Rujira, Liu Sicheng, Imamichi Shoji, Sharma Mukesh Kumar, Matsumoto Yoshihisa
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, Tokyo 152-8550, Japan.
Biochem Biophys Res Commun. 2015 Jun 12;461(4):687-94. doi: 10.1016/j.bbrc.2015.04.093. Epub 2015 Apr 28.
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.
XRCC4和DNA连接酶IV（LIG4）在DNA双链断裂（DSB）修复的最后一步通过非同源末端连接（NHEJ）协同作用连接两个DNA末端。然而，这些蛋白质如何定位于细胞核尚未完全清楚。在此，我们构建了XRCC4（K271R）突变体，因为赖氨酸271位于假定的核定位信号（NLS）内，还构建了XRCC4（K210R）突变体，因为据报道赖氨酸210会发生SUMO化，这与XRCC4的核定位有关。将带有EGFP标签的野生型和突变型XRCC4导入HeLa细胞，其中内源性XRCC4已使用针对3'非翻译区的siRNA敲低，并通过荧光显微镜检测其核定位功能。XRCC4（K271R）自身和LIG4的核定位存在缺陷，而XRCC4（K210R）与LIG4的核定位功能正常。为了检测DSB修复功能，将野生型和突变型XRCC4导入XRCC4缺陷的M10细胞。与M10-XRCC4（WT）相比，M10-XRCC4（K271R）在2 Gy γ射线照射后存活分数显著降低，而M10-XRCC4（K210R）则无明显变化。在2 Gy γ射线照射2小时后，M10-XRCC4（K271R）中残留的γ-H2AX焦点数量显著多于M10-XRCC4（WT），而与M10-XRCC4（WT）相比，M10-XRCC4（K210R）中仅略有增加。目前的结果共同表明，XRCC4的赖氨酸271而非赖氨酸210是XRCC4和LIG4核定位所必需的，并且核定位能力对于XRCC4的DSB修复功能至关重要。
