The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions

Bacterial non-homologous end joining (NHEJ) is a key DNA double-strand break (DSB) repair pathway that relies on the Ku-LigD complex to alleviate genomic instability. The Mycobacterium tuberculosis Ku C-terminal region has previously been highlighted for its role in both LigD recruitment to DNA DSBs and stimulation of LigD ligase activity. However, it remains unclear how the Ku C-terminus interacts with and potentially influences enzymatic activities of LigD. Here, we combine NMR spectroscopy, structural modelling and mutational analysis to define the interaction interface between the Ku C-terminal region and LigD. We identify critical residues in Ku (E246, V248, S258, K260, and N266) and the LigD polymerase (D162, V194, R198) and ligase (D522, K579, L580) domains that mediate this interaction. In addition, we address how this interaction influences LigD activity. Functional assays reveal that Ku stimulates LigD ligase activity through contacts with both enzymatic domains, while Ku attenuates template-dependent polymerase activity, contrasting from previous studies in Pseudomonas aeruginosa, potentially through restricting active site accessibility or altering the conformational dynamics of the enzyme. Disrupting the Ku-LigD interface, either through Ku or LigD mutations, abolishes ligase stimulation and restores polymerase activity, highlighting a dual regulatory mechanism. Our data supports a model in which the Ku C-terminal region forms a bipartite interface with LigD to balance repair activity and fidelity. These findings provide mechanistic insight into the Ku-LigD repair mechanism and uncovers species-specific differences in bacterial NHEJ.