Assembly of KU and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at DNA double strand breaks (DSBs) forms DNA-PK holoenzyme as a critical initiating step for non-homologous end joining (NHEJ) repair of DSBs produced by radiation and chemotherapies. Advanced cryo-electron microscopy (cryo-EM) imaging together with breakthrough macromolecular X-ray crystal (MX) structures of KU and DNA-PKcs recently enabled visualization of the ∼600 kDa DNA-PK assembly at near atomic resolution. These important static structures provide the foundation for definition and interpretation of functional movements crucial to mechanistic understanding that can be tested through solution state structure analysis. We herein therefore leverage Cryo-EM and MX structures for the interpretation of synchrotron small-angle X-ray scattering (SAXS) data on DNA-PK conformations in solution to inform the structural mechanism for NHEJ initiation. SAXS, which measures thermodynamic solution-state conformational states and assemblies outside of cryo- and solid-state conditions, unveils the inherent flexibility of KU, DNA-PKcs and DNA-PK. The combined structural measurements reveal mobility of KU80 C-terminal region (KU80CTR), motion/plasticity of HEAT (DNA-PKcs Huntingtin, Elongation Factor 3, PP2 A, and TOR1) regions, allosteric switching upon DNA-PKcs autophosphorylation, and dimeric arrangements of DNA-PK assembly. Importantly, the results uncover displacement of the N-terminal HEAT domain during autophosphorylation as suitable for a regulated release mechanism of DNA-PKcs from DNA-PK to control unproductive access to toxic and mutagenic DNA repair intermediates. These integrated analyses show that the marriage of SAXS with cryo-EM leverages the strengths of both techniques to enable assessment of functional conformations and flexibility defining atomic-resolution molecular mechanisms for DSB repair.

在 DNA 双链断裂 (DSB) 处组装 KU 和 DNA 依赖性蛋白激酶催化亚基 (DNA-PKcs) 形成 DNA-PK 全酶，这是对辐射产生的 DSB 进行非同源末端连接 (NHEJ) 修复的关键起始步骤。化疗。先进的低温电子显微镜 (cryo-EM) 成像以及 KU 和 DNA-PKcs 的突破性大分子 X 射线晶体 (MX) 结构最近使 ∼600 kDa DNA-PK 组装体在接近原子分辨率下的可视化成为可能。这些重要的静态结构为定义和解释对机械理解至关重要的功能运动提供了基础，可以通过解决方案状态结构分析进行测试。因此，我们在此利用 Cryo-EM 和 MX 结构来解释溶液中 DNA-PK 构象的同步加速器小角 X 射线散射 (SAXS) 数据，以告知 NHEJ 起始的结构机制。SAXS 测量低温和固态条件之外的热力学溶液状态构象状态和组装，揭示了 KU、DNA-PKcs 和 DNA-PK 的固有灵活性。组合结构测量揭示了 KU80 C 末端区域 (KU80CTR) 的移动性、HEAT (DNA-PKcs Huntingtin、延伸因子 3、PP2 A 和 TOR1) 区域的运动/可塑性、DNA-PKcs 自磷酸化的变构转换和二聚体排列DNA-PK 组装。重要的，结果揭示了自磷酸化过程中 N 末端 HEAT 结构域的置换适合于 DNA-PK 的 DNA-PKcs 的调节释放机制，以控制对有毒和诱变 DNA 修复中间体的非生产性访问。这些综合分析表明，SAXS 与 cryo-EM 的结合利用了两种技术的优势，能够评估功能构象和定义 DSB 修复的原子分辨率分子机制的灵活性。