Unraveling the role of UvrD1 in bacterial DNA double-strand break repair

Abstract

DNA double-strand breaks are the most lethal form of DNA damage, requiring rapid repair to prevent cell death and maintain genomic integrity. Certain bacteria can bypass double-strand breaks and evade cell death through non-homologous end joining (NHEJ), an error-prone repair pathway dependent on the DNA end-binding protein Ku and the multi-functional ligase LigD. The DNA unwinding helicase UvrD1 from Mycobacterium tuberculosis also participates in NHEJ, with Ku enhancing its activity. However, the mechanistic role of the Ku-UvrD1 complex in mycobacterial NHEJ remains unknown. This thesis investigates the molecular mechanisms and key protein-protein interactions required for Ku-stimulated UvrD1 helicase activity in response to DNA damage. I have found that the Ku C-terminus, unique to bacteria, is crucial for stimulating and regulating UvrD1 helicase activity. UvrD1 binds Ku with moderate affinity, but loss of the Ku C-terminal region decreases this affinity. Ku prebound to DNA increases UvrD1 binding affinity, indicating that DNA is necessary for a stable interaction. Structure-guided mutagenesis of UvrD1 identified key amino acids that contribute to roles in DNA binding, and helicase activity, through structural stabilization of the 2B domain. Atomic force microscopy (AFM) revealed that the Ku C-terminus is needed for translocation on the DNA, and for recruiting UvrD1 to the unwinding junction. I have also developed a detailed AFM protocol to optimize imaging conditions and analysis workflow, providing a resource to reduce the barrier to entry for those interested in using AFM. To assess conservation of function, I also examined Pseudomonas aeruginosa UvrD and found that Ku similarly interacts with and stimulates UvrD helicase activity, broadening the role of superfamily1 helicases in bacterial NHEJ. Finally, I found that M. tuberculosis UvrD1 interacts with LigD, further supporting its role in NHEJ. These findings advance the fundamental understanding of bacterial NHEJ, offering potential targets for antimicrobial therapeutics development.