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bioRxiv · 10.64898/2025.12.12.693941

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

Sowa, D. J.·Huang, J.·Marwaha, A.·Subramaniam, A.·Doubleday, C.·Warner, M. M.·Byun, J. A.·Melacini, G.·Andres, S. N.
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Abstract

Bacterial non-homologous end joining (NHEJ) is a DNA double-strand break (DSB) repair pathway that relies on the Ku-LigD complex to alleviate genomic instability. The Mycobacterium tuberculosis Ku C-terminus has been highlighted for its role in LigD recruitment to DNA DSBs and stimulation of ligase activity. However, it remains unclear how the Ku C-terminus interacts with and potentially influences other LigD activities. Here, we combine NMR spectroscopy, structural modelling and mutational analysis to define the interaction interface between the Ku C-terminus 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. Functional assays reveal that Ku stimulates LigD ligase activity through contacts with both polymerase and ligase domains, while Ku attenuates template-dependent polymerase activity, contrasting previous studies with Pseudomonas aeruginosa homologs. 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 where Kus C-terminal region forms a bipartite interface with LigD to balance repair activity. These findings provide mechanistic insight into the Ku-LigD repair mechanism and uncovers species-specific differences in bacterial NHEJ. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/693941v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@802e3org.highwire.dtl.DTLVardef@1f5ac14org.highwire.dtl.DTLVardef@1f46677org.highwire.dtl.DTLVardef@7312b5_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Sowa, D. J., Huang, J., Marwaha, A., Subramaniam, A., Doubleday, C., Warner, M. M., Byun, J. A., Melacini, G., Andres, S. N.. 2025-12-14. The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions. https://doi.org/10.64898/2025.12.12.693941

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