The active DNA-PK holoenzyme occupies a tensed state in a staggered synaptic complex

• Published in: Structure (London) 2021-05, Vol.29 (5), p.467-478.e6
• Main Authors: Hepburn, Morgan, Saltzberg, Daniel J., Lee, Linda, Fang, Shujuan, Atkinson, Claire, Strynadka, Natalie C.J., Sali, Andrej, Lees-Miller, Susan P., Schriemer, David C.
• Format: Article
• Language: English
• Subjects: Binding Sites; crosslinking mass spectrometry; DNA End-Joining Repair; DNA repair; DNA-Activated Protein Kinase - chemistry; DNA-Activated Protein Kinase - metabolism; DNA-PK; HeLa Cells; Holoenzymes; Humans; hydrogen/deuterium exchange; modeling; Molecular Docking Simulation; non-homologous end-joining; Protein Binding; structure; synaptic complex; Synaptonemal Complex - chemistry; Synaptonemal Complex - metabolism
• ISSN: 0969-2126; 1878-4186
• DOI: 10.1016/j.str.2020.12.006

In the non-homologous end-joining (NHEJ) of a DNA double-strand break, DNA ends are bound and protected by DNA-PK, which synapses across the break to tether the broken ends and initiate repair. There is little clarity surrounding the nature of the synaptic complex and the mechanism governing the transition to repair. We report an integrative structure of the synaptic complex at a precision of 13.5 Å, revealing a symmetric head-to-head arrangement with a large offset in the DNA ends and an extensive end-protection mechanism involving a previously uncharacterized plug domain. Hydrogen/deuterium exchange mass spectrometry identifies an allosteric pathway connecting DNA end-binding with the kinase domain that places DNA-PK under tension in the kinase-active state. We present a model for the transition from end-protection to repair, where the synaptic complex supports hierarchical processing of the ends and scaffold assembly, requiring displacement of the catalytic subunit and tension release through kinase activity. [Display omitted] •The synaptic complex formed by DNA-PK adopts a staggered “head-to-head” arrangement•A plug domain functions to block the end of the double-strand DNA•When bound to DNA and primed with nucleotide, the holoenzyme adopts a tensed state•The geometry supports the assembly of scaffolding factor to manage repair transitions Hepburn et al. used an integrative approach relying on mass spectrometry to demonstrate that the massive trimeric complex DNA-PK caps DNA double-strand breaks with a strongly protective plug-like domain in a large, unstable synaptic structure that holds the two ends far apart.