Cryo-EM structure of human DNA-PK holoenzyme

• Published in: Cell research Vol. 27; no. 11; pp. 1341 - 1350
• Main Authors: Yin, Xiaotong, Liu, Mengjie, Tian, Yuan, Wang, Jiawei, Xu, Yanhui
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.11.2017; Nature Publishing Group
• Subjects: 631/1647/2258/1258/1259; 631/337/1427; 631/45/607/1163; 631/45/607/275; Activation; Allosteric properties; Allosteric Regulation; Biomedical and Life Sciences; Catalysis; Cell Biology; Cryoelectron Microscopy; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA Breaks, Double-Stranded; DNA repair; DNA structure; DNA-Activated Protein Kinase - chemistry; DNA-Activated Protein Kinase - metabolism; DNA-dependent protein kinase; Electron microscopy; Holoenzymes - chemistry; Homology; Humans; Kinases; Ku Autoantigen - chemistry; Life Sciences; Models, Molecular; Non-homologous end joining; Nuclear Proteins - chemistry; Nuclear Proteins - metabolism; Original; original-article; Protein kinase C; Protein-serine/threonine kinase; DNA-PKcs; DNA-PK; Cryo-EM structure; NHEJ; activation
• ISSN: 1001-0602; 1748-7838; 1748-7838
• DOI: 10.1038/cr.2017.110

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 A 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 in- ward 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 activa- tion 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 mecha- nism for recognition and protection of broken DNA ends and provide a structural basis for understanding the activa- tion of DNA-PKcs and DNA-PK-mediated NHEJ pathway.