How a Mycobacterium protein steers DNA repair

DNA double-strand breaks (DSBs) are among the most dangerous forms of genetic damage because they can destabilize a genome if not repaired. Bacteria cope with these breaks using a pathway called non-homologous end joining (NHEJ), which relies on a partnership between two proteins, Ku and LigD, to patch broken DNA ends. In Mycobacterium tuberculosis, the tail end of Ku — its C-terminus — has been known to help recruit LigD to breaks and boost LigD’s ligase activity, but researchers could not yet explain how that region contacts LigD or whether it affects other LigD functions. Led by corresponding author Sara N. Andres, the team set out to map the interaction between the Ku C-terminus and LigD and to test what those contacts do to LigD’s multiple activities. The work aims to move beyond the idea of a simple recruitment role and to reveal whether Ku actively guides which repair reactions LigD performs at a break. By focusing on this specific protein interface, the researchers sought mechanistic detail that had been missing from earlier, broader descriptions of bacterial NHEJ.

To define how Ku and LigD touch and influence one another, the authors used a combination of NMR spectroscopy, structural modelling and mutational analysis. These approaches allowed them to identify precise amino-acid residues that mediate the interaction: in Ku, the critical positions were E246, V248, S258, K260 and N266; in the LigD polymerase domain the key residues were D162, V194 and R198; and in the LigD ligase domain they found D522, K579 and L580. Functional assays showed that Ku stimulates LigD’s ligase activity by contacting both the polymerase and ligase domains, while at the same time Ku reduces template-dependent polymerase activity — a contrast to prior observations in Pseudomonas aeruginosa homologs. When the team disrupted the Ku–LigD interface by mutating either Ku or LigD at those critical sites, ligase stimulation was lost and polymerase activity returned. Taken together, the data support a model in which the Ku C-terminal region forms a bipartite interface with LigD to balance different repair activities.

The study sheds mechanistic light on how the Ku–LigD complex orchestrates repair choices at DNA double-strand breaks in Mycobacterium tuberculosis. Rather than acting simply as a recruiter or passive scaffold, Ku’s C-terminus appears to be an active regulator that both promotes ligation and suppresses template-dependent polymerization, flipping the balance of LigD activities depending on contact points. The finding of a bipartite interface clarifies why disrupting those contacts has predictable functional consequences: loss of ligase stimulation and restoration of polymerase function. Importantly, the work highlights species-specific differences in bacterial NHEJ by contrasting the Mycobacterium tuberculosis behavior with that seen in Pseudomonas aeruginosa homologs, suggesting that NHEJ mechanisms can vary across bacteria. These insights provide a more detailed view of bacterial DNA repair machinery and offer defined molecular targets for future basic research into how pathogens maintain genome stability under stress.