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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI064693-09
Application #
8461280
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Lacourciere, Karen A
Project Start
2010-05-15
Project End
2014-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
9
Fiscal Year
2013
Total Cost
$497,253
Indirect Cost
$236,775
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
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
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
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

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