Mycobacteria are a genus of the phylum Actinobacteria that includes the human pathogens M. tuberculosis and M. leprae and their avirulent relative M. smegmatis. Human infections with M. tuberculosis (the agent of tuberculosis) and M. leprae (agent of leprosy) cause substantial human suffering. Tuberculosis accounts for ~2 million deaths annually. M. tuberculosis is increasingly antibiotic resistant, solely through the acquisition of mutations in chromosomal genes. The importance of mutagenesis in the evolution of M. tuberculosis antimicrobial resistance, and the related importance of DNA repair pathways in resisting host-inflicted DNA damage, prompts our interest in the mechanisms by which mycobacteria respond to and repair DNA damage. In addition to this important relevance to human health, mycobacteria have emerged as fertile model system to study prokaryotic DNA repair, due to the complexity of the pathways involved and the novel mechanisms that govern these pathways. Our long range goals in this project are to elucidate the DNA repair mechanisms of Mycobacteria. Our work supported by this award has shown that mycobacterial DNA repair differs from the classic E. coli model system with respect to the number of pathway options for the repair of DNA double-strand breaks (DSBs), the roster of DNA repair enzymes, and the regulation of the DNA damage response (DDR). Our premise is that understanding the distinctive features of mycobacterial DNA repair will illuminate the evolution and diversification of repair strategies and suggest new approaches to combat mycobacterial infection and emergence of antibiotic resistance. Our agenda for the next phase of the project focuses on three themes in mycobacterial DNA repair: (i) the mechanism of DSB resection during homologous recombination (HR) by the AdnAB helicase-nuclease; (ii) the role of RecA phosphorylation and RecA interaction with membrane phospholipids in controlling the DNA damage response; and (iii) the structure and repair activities of the DNA helicase Lhr. We propose a combined approach that leverages integrated biochemical, structural, and genetic approaches to understand these DNA repair systems of mycobacteria. Through these studies we will elucidate new mechanisms of DNA repair in mycobacteria, discoveries that will both advance basic knowledge of prokaryotic DNA repair and elucidate pathways relevant to M. tuberculosis drug resistance and pathogenesis.

Public Health Relevance

This project seeks to understand the pathways and mechanisms of DNA repair and mutagenesis in mycobacteria. These studies will both elucidate basic mechanisms of DNA repair in bacteria, but also inform our understanding of how mycobacterial pathogens, including M. tuberculosis, the causative agent of the disease tuberculosis, evolve antibiotic resistance. The long term goal of these studies is, through basic understanding of mycobacterial DNA repair pathways, to validate DNA repair as a therapeutic target for mycobacterial infections, thereby advancing efforts to control tuberculosis and other mycobacterial infections.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Mendez, Susana
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Sloan-Kettering Institute for Cancer Research
New York
United States
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Ejaz, Anam; Shuman, Stewart (2018) Characterization of Lhr-Core DNA helicase and manganese- dependent DNA nuclease components of a bacterial gene cluster encoding nucleic acid repair enzymes. J Biol Chem 293:17491-17504
Wipperman, Matthew F; Heaton, Brook E; Nautiyal, Astha et al. (2018) Mycobacterial Mutagenesis and Drug Resistance Are Controlled by Phosphorylation- and Cardiolipin-Mediated Inhibition of the RecA Coprotease. Mol Cell 72:152-161.e7
Ejaz, Anam; Ordonez, Heather; Jacewicz, Agata et al. (2018) Structure of mycobacterial 3'-to-5' RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases. Nucleic Acids Res 46:442-455
Uson, Maria Loressa; Carl, Ayala; Goldgur, Yehuda et al. (2018) Crystal structure and mutational analysis of Mycobacterium smegmatis FenA highlight active site amino acids and three metal ions essential for flap endonuclease and 5' exonuclease activities. Nucleic Acids Res 46:4164-4175
Gupta, Richa; Unciuleac, Mihaela-Carmen; Shuman, Stewart et al. (2017) Homologous recombination mediated by the mycobacterial AdnAB helicase without end resection by the AdnAB nucleases. Nucleic Acids Res 45:762-774
Uson, Maria Loressa; Ghosh, Shreya; Shuman, Stewart (2017) The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5' Flaps and the Removal of 5' Adenylylated Products of Aborted Nick Ligation. J Bacteriol 199:
Gupta, Richa; Chatterjee, Debashree; Glickman, Michael S et al. (2017) Division of labor among Mycobacterium smegmatis RNase H enzymes: RNase H1 activity of RnhA or RnhC is essential for growth whereas RnhB and RnhA guard against killing by hydrogen peroxide in stationary phase. Nucleic Acids Res 45:1-14
Unciuleac, Mihaela-Carmen; Smith, Paul C; Shuman, Stewart (2016) Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858). J Bacteriol 198:1521-33
Gupta, Richa; Shuman, Stewart; Glickman, Michael S (2015) RecF and RecR Play Critical Roles in the Homologous Recombination and Single-Strand Annealing Pathways of Mycobacteria. J Bacteriol 197:3121-32
Uson, Maria Loressa; Ordonez, Heather; Shuman, Stewart (2015) Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3'-to-5' Helicase That Unwinds 3'-Tailed RNA Duplexes and RNA:DNA Hybrids. J Bacteriol 197:3057-65

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