Non-homologous end joining (NHEJ) is one of two critical mechanisms utilized in humans to repair DNA double-strand breaks (DSBs). Unrepaired or incorrect repair of DSBs can lead to apoptosis or cancer. NHEJ involves several proteins, including the Ku70/80 heterodimer, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), X-ray cross-complementing protein 4 (XRCC4), XRCC4-like factor (XLF), and ligase IV. These core proteins bind DSBs and ligate the damaged DNA ends. However, details of the structural assembly of these proteins remain unclear. Here, we present cryo-EM structures of NHEJ supercomplexes that are composed of these core proteins and DNA, revealing the detailed structural architecture of this assembly. We describe monomeric and dimeric forms of this supercomplex and also propose the existence of alternate dimeric forms of long-range synaptic complexes. Finally, we show that mutational disruption of several structural features within these NHEJ complexes negatively affects DNA repair.

非同源末端连接 (NHEJ) 是人类用于修复 DNA 双链断裂 (DSB) 的两种关键机制之一。DSB 未修复或修复不正确会导致细胞凋亡或癌症。NHEJ 涉及多种蛋白质，包括 Ku70/80 异二聚体、DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs)、X 射线交叉互补蛋白 4 (XRCC4)、XRCC4 样因子 (XLF) 和连接酶 IV。这些核心蛋白结合 DSB 并连接受损的 DNA 末端。然而，这些蛋白质的结构组装细节仍不清楚。在这里，我们展示了由这些核心蛋白和 DNA 组成的 NHEJ 超复合物的冷冻电镜结构，揭示了该组装体的详细结构。我们描述了这种超复合体的单体和二聚体形式，还提出了远程突触复合体的交替二聚体形式的存在。最后，我们表明这些 NHEJ 复合体中的几个结构特征的突变破坏会对 DNA 修复产生负面影响。