Oligomerisation of Ku from Mycobacterium tuberculosis promotes DNA synapsis

Abstract Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB), is estimated to infect nearly one-quarter of the global population. A key factor in its resilience and persistence is its robust DNA repair capacity. Non-homologous end joining (NHEJ) is the primary pathway for repairing DNA double-strand breaks (DSBs) in many organisms, including Mtb , where it is mediated by the Ku protein and the multifunctional LigD enzyme. In this study, we demonstrate that Ku is essential for mycobacterial survival under DNA-damaging conditions. Using cryogenic electron microscopy (cryo-EM), we solved high-resolution structures of both the apo and DNA-bound forms of the Ku- Mtb homodimer. Our structural and biophysical analyses reveal that Ku forms an extended proteo-filament upon binding DNA. We identify critical residues involved in filament formation and DNA synapsis and show that their mutation severely impairs bacterial viability. Furthermore, we propose a model in which the C-terminus of Ku regulates DNA binding and loading and facilitates subsequent recruitment of LigD. These findings provide unique insights into bacterial DNA repair and guide future therapeutics.