Volume 27, No 11, Nov 2017

ISSN: 1001-0602 
EISSN: 1748-7838 2018 
impact factor 17.848* 
(Clarivate Analytics, 2019)

Volume 27 Issue 11, November 2017: 1341-1350

ORIGINAL ARTICLES

Cryo-EM structure of human DNA-PK holoenzyme

Xiaotong Yin^1,2,3,*, Mengjie Liu^1,2,3,*, Yuan Tian^1,2,3, Jiawei Wang⁴ and Yanhui Xu^1,2,3,5

¹Fudan University Shanghai Cancer Center, Institute of Biomedical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China
²Key Laboratory of Molecular Medicine, Ministry of Education, Department of Systems Biology for Medicine, School of Basic Medical Sciences, Shanghai Medical College of Fudan University, Shanghai 200032, China
³State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, School of Life Sciences, Fudan University, Shanghai 200433, China
⁴State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China
⁵CAS Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China Correspondence: Yanhui Xu,(xuyh@fudan.edu.cn)

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.

10.1038/cr.2017.110

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