Tuberculosis bacteria use just two proteins to repair broken DNA, while human cells need a dozen or more—and researchers want to know how that stripped-down repair machine works. This matters because *Mycobacterium tuberculosis* infects roughly a quarter of the world’s population and can survive inside human lungs for decades. The bacterium’s ability to fix DNA damage is essential for its long-term survival, yet the molecular structure of its repair machinery has remained largely unknown. Without knowing how the two key proteins—Ku and LigD—fit together and interact with damaged DNA, scientists cannot design drugs that deliberately break that repair system. If this project succeeds, it will produce the first complete structural images of the bacterial DNA-repair complex, using cryo-electron microscopy. Those structures could reveal weak points that future antimicrobials might target. The work is fundamentally curiosity-driven: understanding how a minimal repair system evolved from bacteria to humans. But similar fundamental studies of bacterial DNA repair have previously led directly to antibiotic development, and a clearer picture of how Mtb keeps its genome intact could open a new route for drugs against a pathogen that kills over a million people each year.
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In all kingdoms of life, DNA damaging agents can jeopardize the integrity of the cellular genome, resulting in a range of developmental defects. A break across both strands of DNA is the most critical form of DNA damage and if left unrepaired can ultimately result in cell death. Non-homologous end joining (NHEJ) is considered to be the major repair pathway of DNA-double strand breaks (DSBs) in all organisms. Due to the advancement in the technology and methodology associated with cryo-electron microscopy (Cryo-EM) in the past few years, the molecular understanding of NHEJ in humans has significantly advanced. However, although NHEJ in bacteria is a simplified system utilising fewer protein components, the bacterial NHEJ mechanism and protein interplay are not well understood. The intracellular human pathogen Mycobacterium tuberculosis (Mtb) causes the respiratory disease Tuberculosis (TB), which affects one quarter of the world population and in some cases the bacteria may survive in infected humans for decades. Understanding the fundamental process of DNA-repair in Mtb will help us to determine the importance for the bacteria to survive and cause infection, and as a potential target for future antimicrobials. The NHEJ mechanism in Mtb involves Ku and the multifunctional protein LigD. Studies have shown that Ku and LigD directly interact in order to repair DSBs. However, there has been limited structural information, with only separate domains of LigD being solved through X-ray crystallography and no full structures or the proteins in complex. The structural and functional studies we propose will help understand how only Ku and LigD can fulfil the multiple roles of NHEJ that are fulfilled by multiple different proteins in humans and will provide mechanistic insights into how this system has evolved from prokaryotes to higher eukaryotes. Within this proposal, we aim to utilise cryo-EM to solve structures of Ku and LigD in complex with various DNA substrates. We will also use single-molecule studies, site-directed mutagenesis and DNA repair assays to understand the dynamics of the NHEJ mechanism and the importance of key interacting protein residues within the structures we are able to determine. Furthermore, we will also evaluate the importance of these NHEJ proteins within Mtb and our findings from structural work will be further validated in complementation studies using Mtb, Mycobacterium smegmatis and Mycobacterium marinum. Finally, we will investigate phosphorylation of Ku and LigD and their importance for specific structural interactions and will uncover specific signalling pathways controlling formation of the NHEJ DNA-repair complexes. Overall, we will determine the precise mechanism of NHEJ in Mtb, which will have huge benefit for understanding the mechanism of NHEJ in bacteria, and other organism, and lead to potential development of novel antimicrobials against Mtb.
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