DNA double-stranded breaks (DSBs) pose a formidable threat to genomic stability, with the potential to disrupt cellular processes and trigger cell death if left unrepaired. Mycobacterium tuberculosis, the causative agent of tuberculosis, strategically evades the host immune system by entering a dormant state within immune cells, particularly macrophages. The pathogen's ability to endure extended dormancy underscores its remarkable resistance to both host defences and antibiotic treatments. In this context, the DNA double-stranded break repair machinery emerges as a vital component for M. tuberculosis to counteract genotoxic stress during dormancy.
This study delves into the intricate role of the DNA-binding protein Ku, a pivotal initiator of the prokaryotic DSB repair process. Using a comprehensive multidisciplinary approach involving cryo-electron microscopy, hydrogen-deuterium exchange mass spectrometry, and fluorescence resonance energy transfer, we provide unprecedented insights into the initial stages and dynamics of prokaryotic DSB repair. Our findings unveil the first atomic structure of the prokaryotic DSB repair protein Ku in complex with DNA, elucidating Ku-mediated DNA synapsis. Remarkably, mycobacterial Ku demonstrates a unique ability to form higher-order oligomers exclusively in the presence of DNA, facilitated by interactions between adjacent Ku homodimers. Our study uncovers that Ku plays a critical and sufficient role in prokaryotic synapsis, bringing two broken DNA ends into proximity through its capacity to form higher-order oligomers. Additionally, we identify key DNA-protein and protein-protein interactions that are likely crucial for the NHEJ synapsis process.
Understanding the structural dynamics of prokaryotic DSB repair, particularly the role of Ku, sheds light on the mechanisms employed by M. tuberculosis to navigate genotoxic stress and ensure survival during dormancy. This knowledge may open avenues for the development of targeted therapeutic strategies against tuberculosis by disrupting essential repair processes in this resilient pathogen.