In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70–Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis. However, several regions, especially the C-terminal regions (CTRs) of the XRCC4 and XLF scaffolding proteins, have largely remained unresolved in experimental structures, which hampers the understanding of their functions. Here we used magnetic resonance techniques and biochemical assays to comprehensively characterize the interactions and dynamics of the XRCC4 and XLF CTRs at residue resolution. We show that the CTRs of XRCC4 and XLF are intrinsically disordered and form a network of multivalent heterotypic and homotypic interactions that promotes robust cellular NHEJ activity. Importantly, we demonstrate that the multivalent interactions of these CTRs lead to the formation of XLF and X4L4 condensates in vitro, which can recruit relevant effectors and critically stimulate DNA end ligation. Our work highlights the role of disordered regions in the mechanism and dynamics of NHEJ and lays the groundwork for the investigation of NHEJ protein disorder and its associated condensates inside cells with implications in cancer biology, immunology and the development of genome-editing strategies.

在哺乳动物细胞中，DNA 双链断裂主要通过非同源末端连接 (NHEJ) 进行修复。在修复过程中，Ku70-Ku80 异二聚体 (Ku)、X 射线修复交叉互补 4 (XRCC4) 与 DNA 连接酶 4 (X4L4) 和 XRCC4 样因子 (XLF) 形成复合物，形成灵活的支架，将断裂的 DNA 末端固定在一起。最近通过单颗粒冷冻电子显微镜分析获得了对 NHEJ 支架的结构组织及其 DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 调节的深入了解。然而，实验结构中的几个区域，尤其是 XRCC4 和 XLF 支架蛋白的 C 末端区域 (CTR)，在很大程度上仍未得到解决，这阻碍了对其功能的理解。在这里，我们使用磁共振技术和生化测定来全面表征 XRCC4 和 XLF CTR 在残留分辨率下的相互作用和动态。我们发现 XRCC4 和 XLF 的 CTR 本质上是无序的，并形成多价异型和同型相互作用的网络，从而促进强大的细胞 NHEJ 活性。重要的是，我们证明这些 CTR 的多价相互作用导致体外 XLF 和 X4L4 缩合物的形成，这可以招募相关效应子并严重刺激 DNA 末端连接。我们的工作强调了无序区域在 NHEJ 机制和动力学中的作用，并为研究 NHEJ 蛋白无序及其细胞内相关凝聚物奠定了基础，这对癌症生物学、免疫学和基因组编辑策略的发展具有重要意义。