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Peer-reviewedCC BY-NC 4.0Nucleic Acids Research

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

  • Dana J Sowa1,2
  • Jinfeng Huang3
  • Anmol Marwaha1,2
  • Asha Subramaniam1,2
  • Caitlin Doubleday1,2
  • Monica M Warner1,2
  • Jung Ah Byun3
  • Giuseppe Melacini3
  • Sara N Andres1,2
  1. 1Department of Biochemistry and Biomedical Sciences, McMaster University , Hamilton, Ontario L8S 4K1 ,
  2. 2Michael G. DeGroote Institute for Infectious Disease Research, McMaster University , Hamilton, Ontario L8S 4L8 ,
  3. 3Department of Chemistry and Chemical Biology, McMaster University , Hamilton, Ontario L8S 4K1 ,
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Abstract

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-terminus has been highlighted for its role in LigD recruitment to DSBs and stimulation of LigD 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 in 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 Ku’s 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.

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Cite

BibTeXRIS
BibTeX
@article{sowa2026mycobacterium,
  title = {{The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions}},
  author = {Dana J Sowa and Jinfeng Huang and Anmol Marwaha and Asha Subramaniam and Caitlin Doubleday and Monica M Warner and Jung Ah Byun and Giuseppe Melacini and Sara N Andres},
  journal = {Nucleic Acids Research},
  year = {2026},
  month = oct,
  doi = {10.1093/nar/gkag1036},
  url = {https://academic.oup.com/nar/article/doi/10.1093/nar/gkag1036/9004259},
  note = {CC BY-NC 4.0},
}
RIS
TY  - JOUR
TI  - The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions
AU  - Dana J Sowa
AU  - Jinfeng Huang
AU  - Anmol Marwaha
AU  - Asha Subramaniam
AU  - Caitlin Doubleday
AU  - Monica M Warner
AU  - Jung Ah Byun
AU  - Giuseppe Melacini
AU  - Sara N Andres
PY  - 2026
DA  - 2026/10/01
JO  - Nucleic Acids Research
DO  - 10.1093/nar/gkag1036
UR  - https://academic.oup.com/nar/article/doi/10.1093/nar/gkag1036/9004259
AB  - 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-terminus has been highlighted for its role in LigD recruitment to DSBs and stimulation of LigD 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 in 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 Ku’s 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.
SN  - 0305-1048
SN  - 1362-4962
ER  -