Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, United States.
Harvard Graduate Program in Biophysics, Harvard Medical School, Boston, United States.
Elife. 2020 Dec 8;9:e61920. doi: 10.7554/eLife.61920.
Non-homologous end joining (NHEJ) is the predominant pathway that repairs DNA double-strand breaks in vertebrates. During NHEJ DNA ends are held together by a multi-protein synaptic complex until they are ligated. Here, we use egg extract to investigate the role of the intrinsically disordered C-terminal tail of the XRCC4-like factor (XLF), a critical factor in end synapsis. We demonstrate that the XLF tail along with the Ku-binding motif (KBM) at the extreme C-terminus are required for end joining. Although the underlying sequence of the tail can be varied, a minimal tail length is required for NHEJ. Single-molecule FRET experiments that observe end synapsis in real-time show that this defect is due to a failure to closely align DNA ends. Our data supports a model in which a single C-terminal tail tethers XLF to Ku, while allowing XLF to form interactions with XRCC4 that enable synaptic complex formation.
非同源末端连接（NHEJ）是脊椎动物修复 DNA 双链断裂的主要途径。在 NHEJ 过程中，DNA 末端通过一个多蛋白突触复合物保持在一起，直到它们被连接。在这里，我们使用卵提取物来研究 XRCC4 样因子（XLF）的内部分散 C 末端尾巴的作用，XLF 是末端连接的关键因子。我们证明，XLF 尾巴与极端 C 末端的 Ku 结合基序（KBM）一起是末端连接所必需的。尽管尾巴的基础序列可以变化，但 NHEJ 需要最小的尾巴长度。实时观察末端连接的单分子 FRET 实验表明，这种缺陷是由于 DNA 末端未能紧密对齐所致。我们的数据支持这样一种模型，即单个 C 末端尾巴将 XLF 固定在 Ku 上，同时允许 XLF 与 XRCC4 形成相互作用，从而形成突触复合物。
