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.
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.
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