A break in both strands of the chromosomal DNA, termed as the “double-strand break (DSB)”, is particularly lethal and diverse mechanisms of repair exist in different organisms to ensure genome integrity. Mycobacterium includes both pathogenic and non-pathogenic species, and DNA repair genes are highly homologous across the species of this genus. Our recent studies have indicated that mycobacteria, including the human pathogen M. tuberculosis, encode three DSB repair pathways to reseal DSBs and ensure survival: (i) RecA-dependent homologous recombination (HR), (ii) Ku-dependent nonhomologous end joining (NHEJ), and (iii) RecBCD-dependent single-strand annealing (SSA). Several distinctive features of HR repair are evident in mycobacteria compared to other model bacteria, with a multitude of proteins involved. Of special note is the RecO protein, which plays an important dual role in mycobacterial DSB repair by participating in both HR and SSA mechanisms but its exact molecular function is yet unclear. We propose to investigate genetically which activities of this key player are pertinent to its role in vivo. By sequence alignment, we have identified amino acid residues in the RecO protein which when mutated are predicted to disrupt specific domains critical for its activity. Experiments involving construction of domain-specific chromosomal mutants of recO and investigation of the effects of these mutations on mycobacterial DSB repair mechanisms will be presented. The findings of this study are also pertinent for developing new combinatorial therapeutic approaches against mycobacterial pathogens.

Role of RecO in the DNA double-strand break repair mechanisms of Mycobacteria

2019 Spring National Meeting

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