# Bringing the ends together: cryo-EM structures of mycobacterial Ku in complex with DNA define its role in NHEJ synapsis

## TL;DR

This study reveals the structure and function of mycobacterial Ku, a key protein in DNA repair in tuberculosis bacteria, using cryo-EM and other techniques.

## Contribution

The first cryo-EM structures of mycobacterial Ku in DNA-bound and higher-order forms are presented, revealing a unique DNA synapsis mechanism in prokaryotes.

## Key findings

- Cryo-EM structures of mKu in DNA-bound and higher-order forms reveal a prokaryote-specific DNA synapsis mechanism.
- Key interactions for mKu dimerization, DNA binding, and synapsis are identified through cryo-EM and hydrogen–deuterium exchange mass spectrometry.
- Structure-guided mutagenesis and assays identify residues crucial for DNA binding and synaptic assembly in NHEJ.

## Abstract

Non-homologous end joining (NHEJ) is the sole pathway for repairing double-strand breaks in Mycobacterium tuberculosis during dormancy, relying on mycobacterial Ku (mKu) and ligase D, with mKu as the rate-limiting factor. Despite its essential role, the lack of structural information on prokaryotic Ku has hindered understanding of the molecular mechanisms underlying bacterial two-component NHEJ machinery. Here, we present the first cryo-electron microscopy (cryo-EM) structures of mKu in DNA-bound and higher-order supercomplex forms, revealing a Ku-mediated DNA synapsis mechanism unique to prokaryotes. Integrating cryo-EM with hydrogen–deuterium exchange mass spectrometry, we define key mKu–mKu dimerization, DNA-binding, and synapsis interactions essential for efficient NHEJ, bridging structure with function. Structure-guided in silico mutagenesis, coupled with electrophoretic mobility shift assays, identifies residues essential for DNA binding and synaptic assembly, which are crucial for NHEJ. Förster resonance energy transfer confirms DNA-dependent mKu oligomerization in solution, while live-cell imaging captures its spatiotemporal dynamics during double-stranded DNA break repair. These findings provide fundamental insights into the architecture and function of prokaryotic NHEJ, positioning mKu as a potential therapeutic target against tuberculosis and offering a framework for understanding DNA repair across bacterial species.

Graphical Abstract