Department of Biochemistry, University of Cambridge, Cambridge, UK.
CryoEM Facility, Department of Biochemistry, University of Cambridge, Cambridge, UK.
Nat Struct Mol Biol. 2021 Jan;28(1):13-19. doi: 10.1038/s41594-020-00517-x. Epub 2020 Oct 19.
DNA double-strand breaks are the most dangerous type of DNA damage and, if not repaired correctly, can lead to cancer. In humans, Ku70/80 recognizes DNA broken ends and recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form DNA-dependent protein kinase holoenzyme (DNA-PK) in the process of non-homologous end joining (NHEJ). We present a 2.8-Å-resolution cryo-EM structure of DNA-PKcs, allowing precise amino acid sequence registration in regions uninterpreted in previous 4.3-Å X-ray maps. We also report a cryo-EM structure of DNA-PK at 3.5-Å resolution and reveal a dimer mediated by the Ku80 C terminus. Central to dimer formation is a domain swap of the conserved C-terminal helix of Ku80. Our results suggest a new mechanism for NHEJ utilizing a DNA-PK dimer to bring broken DNA ends together. Furthermore, drug inhibition of NHEJ in combination with chemo- and radiotherapy has proved successful, making these models central to structure-based drug targeting efforts.
DNA 双链断裂是最危险的 DNA 损伤类型，如果不能正确修复，可能导致癌症。在人类中，Ku70/80 识别 DNA 断裂末端，并在非同源末端连接 (NHEJ) 过程中招募依赖于 DNA 的蛋白激酶催化亚基 (DNA-PKcs) 形成依赖于 DNA 的蛋白激酶全酶 (DNA-PK)。我们呈现了一个分辨率为 2.8 Å 的 DNA-PKcs 的冷冻电镜结构，允许在以前的 4.3 Å X 射线图谱中未解释的区域进行精确的氨基酸序列注册。我们还报道了一个分辨率为 3.5 Å 的 DNA-PK 的冷冻电镜结构，并揭示了由 Ku80 C 末端介导的二聚体。二聚体形成的核心是 Ku80 保守 C 末端螺旋的结构域交换。我们的结果表明了一种利用 DNA-PK 二聚体将断裂的 DNA 末端聚集在一起的新的 NHEJ 机制。此外，NHEJ 的药物抑制与化疗和放疗相结合已被证明是成功的，这使得这些模型成为基于结构的药物靶向努力的核心。
