KU AND LIGD IN BACTERIAL DOUBLE-STRAND BREAK REPAIR

Abstract

DNA double-strand breaks (DSBs) represent one of the most severe forms of genomic damage, threatening cell viability and genomic stability. While homologous recombination is the predominant DSB repair pathway in many organisms’, certain bacteria like M. tuberculosis also possess non-homologous end joining (NHEJ), a pathway that enables survival under conditions where homologous templates for repair are unavailable. Bacterial NHEJ relies predominantly on two proteins: Ku, a DNA end binding protein and LigD, a multifunctional enzyme with ligase, polymerase, nuclease and phosphoesterase activities. Although Ku is known to stimulate LigD-mediated DNA ligation, the molecular basis of this stimulation, and whether Ku regulates LigD’s additional enzymatic functions, remains poorly understood. This thesis investigates how Ku regulates LigD activity in M. tuberculosis, with a particular focus on the structurally unique and functionally flexible C-terminal region of Ku. Through an integrated approach, combining biochemical, biophysical and structural analyses, this work demonstrates that the Ku C-terminus serves as a regulatory module that coordinates DNA binding, synapsis and activation of LigD ligase activity. Additionally, Ku was found to slightly attenuate LigD’s polymerase activity, suggesting that there is an allosteric mechanism between Ku and LigD's enzymatic domains that balances processing and ligation during repair. An ambient-temperature crystal structure of the LigD polymerase domain was also solved, providing insight into conformational dynamics that may contribute to this regulation. Collectively, these findings support a model in which the Ku C-terminus functions as a regulatory hub that modulates LigD enzymatic activities to promote efficient and accurate DNA repair. This work expands the current understanding of bacterial NHEJ and highlights novel regulatory features that distinguish it from eukaryotic NHEJ. These insights may inform future efforts to exploit bacterial DNA repair pathways as potential targets for antimicrobial development, particularly in drug-resistant pathogens such as M. tuberculosis.