Our long-range goals in this project are to elucidate the mechanisms of DNA repair and mutagenesis in Mycobacteria, a bacterial genus that includes the human pathogen M. tuberculosis and its avirulent cousin M. smegmatis. Our expectation is that such understanding will suggest new strategies to interdict against mycobacterial infection and the emergence of antibiotic resistance, the latter caused exclusively by acquired chromosomal mutations. Our studies have revealed that mycobacteria elaborate three genetically distinct pathways of Double Strand Break (DSB) repair: homologous recombination (HR), non-homologous end joining (NHEJ), and single- strand annealing (SSA). Whereas HR is faithful, NHEJ and SSA are liberally mutagenic. In studying the enzymology of these and related pathways, we've focused on the large and diverse rosters of ligases, helicases and polymerases in the mycobacterial proteome. To date, we have characterized the biochemical activities and/or in vivo functions of five mycobacterial DNA ligases (LigA, LigB, LigC1, LigC2 and LigD) and seven mycobacterial helicases, the latter including AdnAB (in HR), RecBCD (in SSA), UvrD1 (in clastogen resistance), UvrD2, SftH, RqlH, and Lhr. M. smegmatis Lhr defines a novel clade of SF2-family helicase with RNA:DNA and DNA:DNA duplex unwinding activities that depend on its distinctive C-terminal domains. Lhr is upregulated in mycobacteria response to DNA damage, but its precise role in repair is unknown. Lhr homologs are found in bacteria from eight different phyla, being especially prevalent in Actinobacteria (including M. tuberculosis) and Proteobacteria. We propose to determine the atomic structure and in vivo function of mycobacterial Lhr. In parallel, we've analyzed the biochemical activities and/or in vivo functions of six mycobacterial polymerases, including LigD-POL (in NHEJ), PolD1, PolD2, DinB1, DinB2 and DinB3. We've shown that LigD- POL, PolD1, PolD2, and DinB2 are unfaithful polymerases and that they readily incorporate ribonucleotides in lieu of deoxyribonucleotides. In the case of DinB2, this ribonucleotide preference reflects the absence of the aromatic steric gate that confers sugar selectivity. DinB2 is also adept at unfaithful incorporation of oxo-dGTP and oxo-rGTP, and at mutagenic bypass of template oxo-dG, indicating a potential role in mutagenesis during oxidative stress. These biochemical properties suggest that DinB2 may be a mediator of chromosomal mutagenesis either when ribonucleotides are abundant or during oxidative stress (or both), a hypothesis that will be investigated in Aim #2. Mycobacteria also manifest a distinctive DNA damage response (DDR) that we have probed by tracking changes in gene expression and post-translational modifications following induction of a single DSB in the bacterial chromosome. We have conducted a SILAC-based proteomic screen for DSB-induced phosphorylation that showed that RecA undergoes phosphorylation at Ser207 subsequent to DSB induction. Ser207 is located in loop 2 of RecA and is adjacent to key residues that interact with ssDNA. Our initial experiments implicate Ser207 phosphorylation as a negative regulator of two distinct RecA functions, DSB repair and SOS induction of mutagenesis, without affecting ssDNA binding or ATPase activity. In the continuation of this award, we propose experiments focused on Lhr, DinB2, and the role of the RecA phosphorylation in HR and the DDR, with the overarching goal of understanding chromosomal mutagenesis and repair in mycobacteria.
Our aims are unified by their focus on genomic integrity and mutagenesis, a focus that is relevant to public health in its relevance to antimicrobial resistance and genome evolution.

Public Health Relevance

This project investigates the DNA repair pathways of mycobacteria, which include the major human pathogen M. tuberculosis, the cause of the disease tuberculosis. Understanding the mechanisms of DNA repair in mycobacteria will provide insight into the origin of mutations in these bacteria, which are the cause of antibiotic resistanc, an increasing problem in both tuberculosis and other bacterial infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI064693-13
Application #
9485824
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Boyce, Jim P
Project Start
2005-02-01
Project End
2020-05-31
Budget Start
2018-06-01
Budget End
2019-05-31
Support Year
13
Fiscal Year
2018
Total Cost
Indirect Cost
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
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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