Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV
Lysine 271 but not lysine 210 of XRCC4 is required for the nuclear localization of XRCC4 and DNA ligase IV
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DOI：
10.1016/j.bbrc.2015.04.093
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发表时间：
2015-06-12
影响因子：
3.1
通讯作者：
Matsumoto, Yoshihisa
中科院分区：
文献类型：
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作者：
Fukuchi, Mikoto;Wanotayan, Rujira;Matsumoto, Yoshihisa
在DNA双链断裂(DSB)修复的最后一步，XRCC4和DNA连接酶IV(LIG4)通过非同源末端连接(NHEJ)共同连接两个DNA末端。然而，目前还不完全清楚这些蛋白质是如何定位在细胞核上的。在这里，我们创建了XRCC4(K271R)突变体，因为Lys271位于可能的核定位信号(NLS)中，以及XRCC4(K210R)突变体，因为据报道Lys210经历了SUMO化，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，利用针对3‘-非翻译区的siRNA将内源性XRCC4基因敲除，并在荧光显微镜下检测其核定位功能。XRCC4(K271R)在自身和LIG4的核定位方面存在缺陷，而XRCC4(K210R)在LIG4的核定位方面是合格的。为了检测DSB修复功能，将野生型和突变型XRCC4导入XRCC4缺失的Mb。与M10-XRCC4(WT)相比，M10-XRCC4(K271R)，而不是M10-XRCC4(K210R)经2Gy射线照射后存活分数显著降低。M10-XRCC4(K271R)照射后2 h残留的γ-H_2AX病灶数显著多于M10-XRCC4(WT)，而M10-XRCC4(K210R)仅略高于M10-XRCC4(WT)。这些结果表明，XRCC4和LIG4的核定位需要的是XRCC4的Lys271而不是Lys210，而核定位能力是XRCC4的DSB修复功能所必需的。(C)2015 Elsevier Inc.保留所有权利。
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 Mb. M10-XRCC4(K271R), but not M10-XRCC4(K210R), showed significantly reduced surviving fraction after 2 Gy gamma-ray irradiation as compared to M10-XRCC4(WT). The number of gamma-H2AX foci remaining 2 h after 2 Gy gamma-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. (C) 2015 Elsevier Inc. All rights reserved.