Nonhomologous end-joining (NHEJ) is a newly appreciated pathway of double strand break repair in bacteria. NHEJ operates in a subset of bacteria, including M. tuberculosis and M. smegmatis, that encode the core NHEJ components: the DNA end-binding protein Ku and ATP- dependent DNA ligase D (LigD). In studies supported by this award, we have characterized the NHEJ pathway in M. smegmatis and M. tuberculosis and demonstrated its dependence on Ku and LigD, with a backup role for ATP-dependent DNA ligase C (LigC). We have shown that repair of 5'overhang and blunt-end double strand breaks (DSBs) by NHEJ is highly mutagenic through the activity of the LigD polymerase domain (LigD-POL), a novel bacterial polymerase that also plays a key structural role in the NHEJ complex. We have developed the homing endonuclease I-SceI for cleavage of the mycobacterial chromosome and shown that NHEJ is required for repair of chromosomal DSBs, a process which also introduces insertions and deletions at repaired ends. Building on this foundation, we now propose an expanded program of biochemical and genetic investigation of the mycobacterial NHEJ pathway, its relationship to other pathways of DSB repair, and its role in M. tuberculosis pathogenesis. By using a newly developed assay of chromosomal DSB repair that discriminates HR, NHEJ and single-strand annealing (SSA) pathways, we will determine the relative frequency of pathway use, molecular outcomes, and effects of DSB end-configuration on DSB repair in wild-type M. smegmatis and mutants deficient in NHEJ components, HR components, or both. Prompted by our findings that UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3'-to-5'DNA helicase, we will probe the role of UvrD1 in DNA repair. We will determine the contribution of RecBCD and the novel mycobacterial helicase/nuclease AdnAB to DNA repair and NHEJ deletion formation through a detailed genetic and biochemical analysis of these enzyme complexes. Finally, we will test whether NHEJ and HR play overlapping roles in M. tuberculosis persistence and latency in the murine model. These studies will provide mechanistic insight into prokaryotic NHEJ and determine the role of NHEJ in pathogenesis, potentially advancing this pathway as a target for antimicrobial development.

Public Health Relevance

This project investigates the novel DNA repair pathway of Nonhomologous end-joining in mycobacteria, including the major human pathogen M. tuberculosis, cause of the disease Tuberculosis. These studies will advance our understanding of how mycobacteria resist elimination by the host and may lead to novel drug strategies for infections caused by mycobacteria.

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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Lacourciere, Karen A
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Sloan-Kettering Institute for Cancer Research
New York
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Gupta, Richa; Unciuleac, Mihaela-Carmen; Shuman, Stewart et al. (2016) Homologous recombination mediated by the mycobacterial AdnAB helicase without end resection by the AdnAB nucleases. Nucleic Acids Res :
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; Chatterjee, Debashree; Glickman, Michael S et al. (2016) 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 :
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
Jacewicz, Agata; Shuman, Stewart (2015) Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains. J Bacteriol 197:2489-98
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
Ordonez, Heather; Uson, Maria Loressa; Shuman, Stewart (2014) Characterization of three mycobacterial DinB (DNA polymerase IV) paralogs highlights DinB2 as naturally adept at ribonucleotide incorporation. Nucleic Acids Res 42:11056-70
Landick, Robert; Krek, Azra; Glickman, Michael S et al. (2014) Genome-Wide Mapping of the Distribution of CarD, RNAP σ(A), and RNAP β on the Mycobacterium smegmatis Chromosome using Chromatin Immunoprecipitation Sequencing. Genom Data 2:110-113
Bhattarai, Hitesh; Gupta, Richa; Glickman, Michael S (2014) DNA ligase C1 mediates the LigD-independent nonhomologous end-joining pathway of Mycobacterium smegmatis. J Bacteriol 196:3366-76
Heaton, Brook E; Barkan, Daniel; Bongiorno, Paola et al. (2014) Deficiency of double-strand DNA break repair does not impair Mycobacterium tuberculosis virulence in multiple animal models of infection. Infect Immun 82:3177-85

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