Journal article

SAP domain forms a flexible part of DNA aperture in Ku70/80

Ales Hnizda, Petr Tesina, Thanh-Binh Nguyen, Zdenek Kukacka, Lukas Kater, Amanda Chaplin, Roland Beckmann, David B Ascher, Petr Novak, Tom L Blundell

FEBS JOURNAL | WILEY | Published : 2021


Nonhomologous end joining (NHEJ) is a DNA repair mechanism that religates double-strand DNA breaks to maintain genomic integrity during the entire cell cycle. The Ku70/80 complex recognizes DNA breaks and serves as an essential hub for recruitment of NHEJ components. Here, we describe intramolecular interactions of the Ku70 C-terminal domain, known as the SAP domain. Using single-particle cryo-electron microscopy, mass spectrometric analysis of intermolecular cross-linking and molecular modelling simulations, we captured variable positions of the SAP domain depending on DNA binding. The first position was localized at the DNA aperture in the Ku70/80 apo form but was not observed in the DNA-b..

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Awarded by Wellcome Trust

Awarded by Jack Brockhoff Foundation

Awarded by National Health, Medical Research Council (NHMRC) of Australia

Awarded by MRC

Awarded by BBSRC

Awarded by European Commission

Awarded by Centre of Molecular Structure Core Facility at BIOCEV

Funding Acknowledgements

AH, DBA and AKC were supported by a Wellcome Trust Investigator Award to TLB (200814/Z/16/Z; 2016). DBA and TBN were funded by the Jack Brockhoff Foundation (JBF 4186, 2016), by an Investigator Grant from the National Health, by Medical Research Council (NHMRC) of Australia (GNT1174405) and in part by the Victorian Government's Operational Infrastructure Support Program. Cryo-electron microscopy data sets were collected in cryo-EM facilities in the Department of Biochemistry, University of Cambridge and the Electron Bio-Imaging Centre Diamond Light Source, proposal EM17057-43 funded by the Wellcome Trust, MRC, and BBSRC. We thank to Dima Chirgadze, Stephen Hardwick, Adriana Klyszejko and Lee Cooper for a kind help with cryo-EM measurements. Mass spectrometry measurements were funded by European Commission H2020 (project EU_FT-ICR_MS - grant agreement ID: 731077) and the Centre of Molecular Structure Core Facility at BIOCEV (LM2018127 CIISB for CMS BIOCEV funded by MEYS CR).