DNA double-strand breaks (DSBs), one of the most cytotoxic forms of DNA damage, can be repaired by the tightly regulated nonhomologous end joining (NHEJ) machinery (Stinson and Loparo and Zhao et al. ). Core NHEJ factors form an initial long-range (LR) synaptic complex that transitions into a DNA-PKcs (DNA-dependent protein kinase, catalytic subunit)–free, short-range state to align the DSB ends (Chen et al. ). Using single-particle cryo–electron microscopy, we have visualized three additional key NHEJ complexes representing different transition states, with DNA-PKcs adopting distinct dimeric conformations within each of them. Upon DNA-PKcs autophosphorylation, the LR complex undergoes a substantial conformational change, with both Ku and DNA-PKcs rotating outward to promote DNA break exposure and DNA-PKcs dissociation. We also captured a dimeric state of catalytically inactive DNA-PKcs, which resembles structures of other PIKK (Phosphatidylinositol 3-kinase-related kinase) family kinases, revealing a model of the full regulatory cycle of DNA-PKcs during NHEJ.

中文翻译：

---

NHEJ 中 DNA-PKcs 结构中间体的冷冻电镜可视化

DNA 双链断裂 (DSB) 是最具细胞毒性的 DNA 损伤形式之一，可以通过严格调控的非同源末端连接 (NHEJ) 机制进行修复（Stinson 和 Loparo 以及等人。）。核心 NHEJ 因子形成初始长程 (LR) 突触复合体，该复合体转变为 DNA-PKcs（DNA 依赖性蛋白激酶，催化亚基）游离的短程状态，以对齐 DSB 末端（Chen等人。）。使用单粒子冷冻电子显微镜，我们观察到了代表不同过渡态的另外三个关键 NHEJ 复合物，其中 DNA-PKc 在每个复合物中采用不同的二聚体构象。DNA-PKcs 自磷酸化后，LR 复合物会发生显着的构象变化，Ku 和 DNA-PKcs 均向外旋转，以促进 DNA 断裂暴露和 DNA-PKcs 解离。我们还捕获了催化失活 DNA-PKcs 的二聚体状态，它类似于其他 PIKK（磷脂酰肌醇 3 激酶相关激酶）家族激酶的结构，揭示了 NHEJ 期间 DNA-PKcs 完整调节周期的模型。