It takes two (Ku) to repair: Mechanistic insights into the minimal NHEJ system of Mycobacterium Tuberculosis

ABSTRACT In this study, we used a combination of structural simulations, ensemble assays and single molecule analysis to shed light on Non-Homologous End-Joining (NHEJ) in Mycobacterium Tuberculosis ( Mtb ), executed by the homodimeric Ku and Ligase D (LigD). We used a monomeric form of the Ku protein to confirm the necessity of homodimerization of Ku to bind DNA. We demonstrated that Mtb Ku and shows limited translocation on DNA and primarily instead stays bound at the DNA ends. The reconstitution of an active Mtb NHEJ machinery, consisting of Ku and LigD, allowed us to characterize the dynamics of each step of the assembly of the Mtb NHEJ machinery at the single molecule level, revealing competition between Ku and LigD for DNA binding. In silico investigations highlighted key residues in Mtb LigD – Ku complex formation. We conducted a mutational analysis of these residues in the polymerase and the ligase domains, respectively. This investigation demonstrated that, although formation of the NHEJ complex is preserved in LigD mutants, mutations in the polymerase domain result in reduced enzymatic activity, whereas mutations in the ligase domain led to its enhancement. Finally, we present a model for NHEJ in M. tuberculosis, where Ku must stay tightly at the DNA ends to properly recruit and regulate LigD enzymatic activity. GRAPHICAL ABSTRACT Mtb Ku and LigD compete for DNA binding, while interactions between Ku and the LigD polymerase (POL) and ligase (LIG) domains differentially modulate enzymatic activity. This image was generated with the assistance of ChatGPT and Gemini and subsequently edited by the authors.