Institute of Clinical Chemistry and Laboratory Diagnostics, Univ Düsseldorf, Medical Faculty, D-40225 Düsseldorf, Germany.
DNA Repair (Amst). 2011 Dec 10;10(12):1232-42. doi: 10.1016/j.dnarep.2011.09.012. Epub 2011 Oct 5.
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.
非同源末端连接（NHEJ）是哺乳动物修复双链 DNA 断裂的主要途径之一。在断裂识别、对齐和处理后，通过必需的 DNA 连接酶 IV 将断裂的 DNA 末端最终重新连接。在细胞中，没有其组成性相互作用伙伴 XRCC4，连接酶 IV 无法发挥功能，当缺失时其变得不稳定，并且假设 XRCC4 也可能需要招募连接酶 IV 到修复位点。为了直接在活细胞中研究两种蛋白质复合物形成的功能，我们在人 HT-1080 细胞克隆中稳定表达它们作为生物荧光融合蛋白。单独表达连接酶 IV 或 XRCC4，或者以大致等摩尔比共表达两者。与内源性表达的蛋白质相比，标记的蛋白质表达水平大大提高。我们表明，过表达的连接酶 IV 仅部分导入细胞核并在那里显示弥散分布，而单独表达的 XRCC4 则完全是核的，明显排除核仁。当连接酶 IV 与 XRCC4 共表达时，两种蛋白质形成天然复合物，连接酶 IV 不仅被有效地导入，而且类似于 XRCC4 的亚核分布。此外，当与 XRCC4 形成复合物时，连接酶 IV 在 XRCC4 有丝分裂细胞分裂后获得了延迟的核再导入。我们进一步通过光漂白确定了在 UVA 激光照射的核位之间在结合状态和扩散状态之间交换的蛋白质动力学。我们发现，在微照射位点处，连接酶 IV/XRCC4 复合物的动态交换率快于单独表达的 XRCC4。总之，我们的发现表明 XRCC4 在控制连接酶 IV 的核输入和亚核分布方面具有新的功能，并表明 XRCC4 调节 Ligase IV/XRCC4 复合物与双链 DNA 断裂处的 NHEJ 机制的动态相互作用。
