Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain.
DNA Repair (Amst). 2009 Dec 3;8(12):1380-9. doi: 10.1016/j.dnarep.2009.09.007.
The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory role in NHEJ. Here, we have purified full length Xrcc4 and the Lig4-Xrcc4 complex, and analysed their structure by single-particle electron microscopy. The three-dimensional structure of Xrcc4 at a resolution of approximately 37A reveals that the C-terminus of Xrcc4 forms a dimeric globular domain connected to the N-terminus by a coiled-coil. The N- and C-terminal domains of Xrcc4 locate at opposite ends of an elongated molecule. The electron microscopy images of the Lig4-Xrcc4 complex were examined by two-dimensional image processing and a double-labelling strategy, identifying the site of the C-terminus of Xrcc4 and the catalytic core of Lig4 within the complex. The catalytic domains of Lig4 were found to be in the vicinity of the N-terminus of Xrcc4. We provide a first sight of the structural organization of the Lig4-Xrcc4 complex, which suggests that the BRCT domains could provide the link of the ligase to Xrcc4 while permitting some movements of the catalytic domains of Lig4. This arrangement may facilitate the ligation of diverse configurations of damaged DNA.
DNA 连接酶 IV-Xrcc4 复合物负责在哺乳动物的 DNA 双链断裂修复的非同源末端连接（NHEJ）途径中连接断裂的 DNA 末端。DNA 连接酶 IV（Lig4）的突变导致人类免疫缺陷和对辐射敏感。只有部分 Lig4 和 Xrcc4 的结构信息可用，而全长蛋白的结构及其在 Lig4-Xrcc4 复合物中的排列方式尚不清楚。Xrcc4 的 C 末端结构域尚未解决，其结构域包含 DNA-PKcs 的磷酸化位点，并且在系统发育上保守，表明在 NHEJ 中具有调节作用。在这里，我们纯化了全长 Xrcc4 和 Lig4-Xrcc4 复合物，并通过单颗粒电子显微镜分析了它们的结构。Xrcc4 的三维结构分辨率约为 37A，揭示了 Xrcc4 的 C 末端形成一个二聚球蛋白形结构域，通过卷曲螺旋与 N 末端连接。Xrcc4 的 N 和 C 末端结构域位于长分子的相对两端。通过二维图像处理和双标记策略检查 Lig4-Xrcc4 复合物的电子显微镜图像，确定了 Xrcc4 的 C 末端和复合物内 Lig4 的催化核心的位置。发现 Lig4 的催化结构域位于 Xrcc4 的 N 末端附近。我们首次观察到 Lig4-Xrcc4 复合物的结构组织，这表明 BRCT 结构域可以为 Ligase 与 Xrcc4 提供连接，同时允许 Lig4 的催化结构域的一些运动。这种排列方式可能有助于连接不同构型的受损 DNA。
