XRCC4 controls nuclear import and distribution of Ligase IV and exchanges faster at damaged DNA in complex with Ligase IV
XRCC4 controls nuclear import and distribution of Ligase IV and exchanges faster at damaged DNA in complex with Ligase IV
复制标题
DOI：
10.1016/j.dnarep.2011.09.012
复制
发表时间：
2011-12-10
期刊：
影响因子：
3.8
通讯作者：
Mielke, Christian
中科院分区：
文献类型：
--
作者：
Berg, Elke;Christensen, Morten O.;Mielke, Christian
非同源末端连接（Non-homologous end-joining， NHEJ）是哺乳动物双链DNA断裂修复的主要途径。经过断裂的识别、排列和加工，断裂的DNA末端最终由必需的DNA连接酶IV重新连接。在细胞中，连接酶IV如果没有其组成的相互作用伙伴XRCC4就无法发挥作用，并且在缺少XRCC4时变得不稳定，并且假设XRCC4也可能是连接酶IV募集到修复位点所必需的。为了研究这两种蛋白在活细胞中直接形成复合物的功能，我们在人HT-1080细胞克隆中稳定地表达了它们作为生物荧光融合蛋白。连接酶IV或XRCC4单独表达或两者以大致等摩尔的比例共表达。与内源性表达蛋白相比，标记蛋白有多种过表达形式。我们发现，过表达的连接酶IV仅部分导入细胞核，并在细胞核中呈扩散分布，而单独表达的XRCC4则完全是细胞核，与核仁明显排斥。当连接酶IV与XRCC4共表达时，两种蛋白形成天然复合物，并且连接酶IV不仅有效导入，而且与XRCC4的亚核分布相似。此外，当连接酶IV与XRCC4复合物时，在XRCC4有丝分裂细胞分裂后获得延迟的核再输入。我们进一步通过光漂白确定了蛋白质在UVA激光照射下在损伤结合态和扩散态之间交换的动力学。我们发现连接酶IV/XRCC4复合物在微辐照位点的动态交换速率比单独表达的XRCC4更快。总之，我们的研究结果证明了XRCC4在控制连接酶IV的核输入和亚核分布方面的新功能，并表明XRCC4调节了连接酶IV/XRCC4复合物在双链DNA断裂时与NHEJ机制的动态相互作用。(C) 2011 Elsevier B.V.版权所有
Non-homologous end-joining (NHEJ) is one major pathway for the repair of double-stranded DNA breaks in mammals. Following break recognition, alignment and processing, broken DNA ends are finally rejoined by the essential DNA Ligase IV. In the cell, Ligase IV is unable to function without its constitutive interaction partner XRCC4 and becomes unstable when it is missing, and it has been assumed that XRCC4 may also be required for recruitment of Ligase IV to repair sites. To investigate the function of complex formation between both proteins directly in the living cell, we stably expressed them as bio-fluorescent fusion proteins in human HT-1080 cell clones. Ligase IV or XRCC4 were expressed either alone or both were co-expressed at a roughly equimolar ratio. Labelled proteins were overexpressed manifold in comparison to endogenously expressed proteins. We show that over-expressed Ligase IV was only partially imported into the nucleus and showed a diffuse distribution there, whereas XRCC4 expressed alone was entirely nuclear with a distinct exclusion from nucleoli. When Ligase IV was co-expressed with XRCC4, both proteins formed the natural complex, and Ligase IV was not only efficiently imported but also resembled the sub-nuclear distribution of XRCC4. In addition, Ligase IV, when in complex with XRCC4, acquired a delayed nuclear reimport after mitotic cell division of XRCC4. We further determined by photobleaching the kinetics with which the proteins exchange at UVA laser-irradiated nuclear sites between damage-bound and diffusing states. We found that the dynamic exchange rate of the Ligase IV/XRCC4 complex at micro-irradiated sites was faster than that of XRCC4 expressed alone. In summary, our findings demonstrate a novel function of XRCC4 in controlling nuclear import and sub-nuclear distribution of Ligase IV, and they suggest that XRCC4 modulates the dynamic interaction of the Ligase IV/XRCC4 complex with the NHEJ machinery at double-stranded DNA breaks. (C) 2011 Elsevier B.V. All rights reserved.