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
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
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
Ryzhikov, Mikhail; Gupta, Richa; Glickman, Michael et al. (2014) RecO protein initiates DNA recombination and strand annealing through two alternative DNA binding mechanisms. J Biol Chem 289:28846-55
Ordonez, Heather; Shuman, Stewart (2014) Mycobacterium smegmatis DinB2 misincorporates deoxyribonucleotides and ribonucleotides during templated synthesis and lesion bypass. Nucleic Acids Res 42:12722-34
Landick, Robert; Krek, Azra; Glickman, Michael S et al. (2014) Genome-Wide Mapping of the Distribution of CarD, RNAP ?(A), and RNAP ? on the Mycobacterium smegmatis Chromosome using Chromatin Immunoprecipitation Sequencing. Genom Data 2:110-113
Unciuleac, Mihaela-Carmen; Shuman, Stewart (2013) Distinctive effects of domain deletions on the manganese-dependent DNA polymerase and DNA phosphorylase activities of Mycobacterium smegmatis polynucleotide phosphorylase. Biochemistry 52:2967-81
Unciuleac, Mihaela-Carmen; Shuman, Stewart (2013) Discrimination of RNA from DNA by polynucleotide phosphorylase. Biochemistry 52:6702-11
Gupta, Richa; Ryzhikov, Mikhail; Koroleva, Olga et al. (2013) A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing. Nucleic Acids Res 41:2284-95
Srivastava, Devendra B; Leon, Katherine; Osmundson, Joseph et al. (2013) Structure and function of CarD, an essential mycobacterial transcription factor. Proc Natl Acad Sci U S A 110:12619-24
Ordonez, Heather; Shuman, Stewart (2013) Mycobacterium smegmatis Lhr Is a DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that prefers to unwind 3'-tailed RNA:DNA hybrids. J Biol Chem 288:14125-34

Showing the most recent 10 out of 27 publications