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.DatabasesEM 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.
Highlights
DNA double-strand breaks represent the most dangerous type of DNA lesions that, if not properly repaired, may compromise genomic integrity of cells
In order to capture the position of the SAP domain in the Ku70/80 heterodimer, we used intermolecular cross-linking followed by single-particle cryo-EM
Using intermolecular cross-linking coupled with cryo-EM and mass spectrometry, we captured the fluctuating position of the SAP domain in Ku70/80
Summary
DNA double-strand breaks represent the most dangerous type of DNA lesions that, if not properly repaired, may compromise genomic integrity of cells. Nonhomologous end joining (NHEJ) is an essential repair mechanism that facilitates religation of DNA breaks throughout the entire cell cycle. This process is mediated by the Ku70/80 complex, which recognizes the ends of the broken DNA and serves as an interaction hub for recruitment of downstream components of the NHEJ pathway [1]. In addition to NHEJ, Ku70/80 plays an important role in multiple biological processes including telomere maintenance [2], HIV replication [3] and suppression of apoptosis [4]. The two subunits share a common domain topology, Abbreviations CTD, C-terminal domain; DSA, Di(N-succinimidyl) adipate; NHEJ, nonhomologous end joining; TF, transcription factor
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