How TB bacteria stitch DNA breaks: cryo-EM reveals Ku's role

Tuberculosis bacteria can survive long periods of dormancy, and during those times they rely on a single pathway to repair dangerous double-strand breaks in their DNA. That pathway, non-homologous end joining or NHEJ, depends on two bacterial proteins: mycobacterial Ku (mKu) and ligase D, with mKu acting as the rate-limiting factor. Until now, a clear picture of how the prokaryotic Ku protein works at the molecular level was missing because no high-resolution structures of bacterial Ku bound to DNA existed. A team led by Isabelle Rouiller set out to fill that gap. They produced the first cryo-EM structures of mycobacterial Ku both bound to DNA and assembled into higher-order super-complexes. Those structures show how Ku molecules come together to align DNA ends — a process called synapsis — and reveal features that are unique to prokaryotic Ku compared with other Ku proteins. By solving these structures, Rouiller and colleagues provided the physical framework needed to understand how the two-component NHEJ machinery in bacteria assembles and functions during DNA repair.

To connect structure with function, the researchers combined cryo-EM with several biochemical and biophysical techniques. They used hydrogen-deuterium exchange mass spectrometry to map regions involved in mKu-mKu dimerization, DNA binding, and synaptic contact points, identifying interaction surfaces critical for forming a repair-competent complex. Structure-guided in silico mutagenesis pinpointed candidate residues predicted to disrupt those interactions, and electrophoretic mobility shift assays then tested how those mutations affected DNA binding and synaptic assembly. The studies showed specific residues are essential for both DNA binding and the higher-order assembly needed for efficient NHEJ. Förster resonance energy transfer experiments confirmed that mKu oligomerization is DNA dependent in solution, demonstrating that the assemblies seen by cryo-EM are not just artifacts of freezing. Finally, live-cell imaging captured the spatiotemporal dynamics of mKu during DSB repair in cells, linking the molecular assemblies to behavior inside living bacteria.

These findings give a first detailed view of the architecture and mechanics of prokaryotic NHEJ, showing how mKu mediates synapsis of DNA ends and how its interactions drive the early steps of repair. By defining the mKu-mKu interfaces, DNA-binding surfaces, and residues required for synaptic assembly, the work bridges structural models with biochemical function, offering concrete targets for further study. Because mKu is rate limiting for NHEJ in Mycobacterium tuberculosis, the new structural and functional map positions mKu as a potential therapeutic target against tuberculosis: small molecules or other interventions that disrupt key interfaces could, in principle, compromise the bacterium's ability to repair double-strand breaks during dormancy. Beyond tuberculosis, the structures provide a framework for understanding DNA repair across bacterial species that use two-component NHEJ, helping researchers explore whether similar mechanisms operate more broadly and how they might be exploited in antimicrobial strategies.