DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase complex composed of a catalytic subunit (DNA-PKcs) and KU70/80 heterodimer bound to DNA. DNA-PK holoenzyme plays a critical role in non-homologous end joining (NHEJ), the major DNA repair pathway. Here, we determined cryo-electron microscopy structure of human DNA-PK holoenzyme at 6.6 Å resolution. In the complex structure, DNA-PKcs, KU70, KU80 and DNA duplex form a 650-kDa heterotetramer with 1:1:1:1 stoichiometry. The N-terminal α-solenoid (∼2 800 residues) of DNA-PKcs adopts a double-ring fold and connects the catalytic core domain of DNA-PKcs and KU70/80-DNA. DNA-PKcs and KU70/80 together form a DNA-binding tunnel, which cradles ∼30-bp DNA and prevents sliding inward of DNA-PKcs along with DNA duplex, suggesting a mechanism by which the broken DNA end is protected from unnecessary processing. Structural and biochemical analyses indicate that KU70/80 and DNA coordinately induce conformational changes of DNA-PKcs and allosterically stimulate its kinase activity. We propose a model for activation of DNA-PKcs in which allosteric signals are generated upon DNA-PK holoenzyme formation and transmitted to the kinase domain through N-terminal HEAT repeats and FAT domain of DNA-PKcs. Our studies suggest a mechanism for recognition and protection of broken DNA ends and provide a structural basis for understanding the activation of DNA-PKcs and DNA-PK-mediated NHEJ pathway.

中文翻译：

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人类DNA-PK全酶的低温EM结构。

DNA依赖性蛋白激酶（DNA-PK）是一种丝氨酸/苏氨酸蛋白激酶复合物，由催化亚基（DNA-PKcs）和与DNA结合的KU70 / 80异二聚体组成。DNA-PK全酶在主要DNA修复途径非同源末端连接（NHEJ）中起关键作用。在这里，我们确定了人类DNA-PK全酶的冷冻电子显微镜结构，分辨率为6.6。在复杂的结构中，DNA-PKcs，KU70，KU80和DNA双链体形成650-kDa异四聚体，化学计量比为1：1：1：1。DNA-PKcs的N端α-电磁素（约2800个残基）具有双环折叠，并连接DNA-PKcs的催化核心结构域和KU70 / 80-DNA。DNA-PKcs和KU70 / 80共同形成DNA结合通道，该通道可形成约30 bp的DNA，并防止DNA-PKcs与DNA双链体一起向内滑动，提出了保护断裂的DNA末端免受不必要的加工的机制。结构和生化分析表明，KU70 / 80和DNA协同诱导DNA-PKcs的构象变化并变构地刺激其激酶活性。我们提出了一种激活DNA-PKcs的模型，其中在DNA-PK全酶形成时产生了变构信号，并通过N-末端HEAT重复序列和DNA-PKcs的FAT域传递到激酶域。我们的研究提出了识别和保护断裂的DNA末端的机制，并为理解DNA-PKcs和DNA-PK介导的NHEJ途径的激活提供了结构基础。结构和生化分析表明，KU70 / 80和DNA协同诱导DNA-PKcs的构象变化并变构地刺激其激酶活性。我们提出了一种激活DNA-PKcs的模型，其中在DNA-PK全酶形成时产生了变构信号，并通过N-末端HEAT重复序列和DNA-PKcs的FAT域传递到激酶域。我们的研究提出了识别和保护断裂的DNA末端的机制，并为理解DNA-PKcs和DNA-PK介导的NHEJ途径的激活提供了结构基础。结构和生化分析表明，KU70 / 80和DNA协同诱导DNA-PKcs的构象变化，并变构地刺激其激酶活性。我们提出了一种激活DNA-PKcs的模型，其中在DNA-PK全酶形成时产生了变构信号，并通过N-末端HEAT重复序列和DNA-PKcs的FAT域传递到激酶域。我们的研究提出了识别和保护断裂的DNA末端的机制，并为理解DNA-PKcs和DNA-PK介导的NHEJ途径的激活提供了结构基础。