SAP domain forms a flexible part of DNA aperture in Ku70/80. (16th February 2021)

• Record Type: Journal Article
• Authors: Hnízda, Aleš; Tesina, Petr; Nguyen, Thanh‐Binh; Kukačka, Zdeněk; Kater, Lukas; Chaplin, Amanda K.; Beckmann, Roland; Ascher, David B.; Novák, Petr; Blundell, Tom L.
• Is Part Of: FEBS journal. Volume 288:Number 14(2021)
• Page Start: 4382
• Page End: 4393
• Publication Date: 2021-02-16
• Subjects: DNA double‐strand break -- integrative structural biology -- Ku70/80 -- nonhomologous end joining -- SAP domain; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15732
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : 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‐bound state. The second position, which was observed in both apo and DNA‐bound states, was found below the DNA aperture, close to the helical arm of Ku70. The localization of the SAP domain in the DNA aperture suggests a function as a flexible entry gate for broken DNA. Databases: EM maps have been deposited in EMDB (EMD‐11933). Coordinates have been deposited in Protein Data Bank (PDB 7AXZ ). Other data are available from corresponding authors upon a request. Abstract : Ku70/80 mediates a recognition of DNA breaks to initiate nonhomologous end joining, an important DNA repair mechanism for maintaining genomic integrity. Our study describes dynamic movements of the SAP domain, a C‐terminal domain in the Ku70 subunit, depending on DNA binding. Using cryo‐EM, mass spectrometry and computational modelling, the SAP domain was localized at the DNA aperture and probably forms a flexible entry gate for broken DNA.