Essential roles for imuA' - and imuB -encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 107; no. 29; pp. 13093 - 13098
• Main Authors: Warner, Digby F., Ndwandwe, Duduzile E., Abrahams, Garth L., Kana, Bavesh D., Machowski, Edith E., Venclovas, Česlovas, Mizrahi, Valerie, Benkovic, Stephen J.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 20-07-2010; National Acad Sciences
• Subjects: Actinomycetes; Amino Acid Motifs; Amino Acid Sequence; Amino acids; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacteriology; Biocatalysis; Biological Sciences; Catalytic Domain; Data processing; Deletion mutant; Deoxyribonucleic acid; DNA; DNA Damage; DNA polymerase; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - metabolism; Drug resistance; Gene deletion; Genetic mutation; Genomes; Immune tolerance; Lesions; Molecular Sequence Data; Mutagenesis; Mutagenesis, Insertional - genetics; Mutation; Mycobacterium tuberculosis; Mycobacterium tuberculosis - enzymology; Operons; Pathogens; Phenotypes; Protein Binding; Protein interaction; Protein Structure, Secondary; Proteins; Structure-Activity Relationship; Truncation
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1002614107

In Mycobacterium tuberculosis (Mtb), damage-induced mutagenesis is dependent on the C-family DNA polymerase, DnaE2. Included with dnaE2 in the Mtb SOS regulon is a putative operon comprising Rv3395c, which encodes a protein of unknown function restricted primarily to actinomycetes, and Rv3394c, which is predicted to encode a Y-family DNA polymerase. These genes were previously identified as components of an imuA-imuB-dnaE2–type mutagenic cassette widespread among bacterial genomes. Here, we confirm that Rv3395c (designated imuA') and Rv3394c (imuB) are individually essential for induced mutagenesis and damage tolerance. Yeast two-hybrid analyses indicate that ImuB interacts with both ImuA' and DnaE2, as well as with the β-clamp. Moreover, disruption of the ImuB-β clamp interaction significantly reduces induced mutagenesis and damage tolerance, phenocopying imuA', imuB, and dnaE2 gene deletion mutants. Despite retaining structural features characteristic of Y-family members, ImuB homologs lack conserved active-site amino acids required for polymerase activity. In contrast, replacement of DnaE2 catalytic residues reproduces the dnaE2 gene deletion phenotype, strongly implying a direct role for the α-subunit in mutagenic lesion bypass. These data implicate differential protein interactions in specialist polymerase function and identify the split imuA'-imuB/dnaE2 cassette as a compelling target for compounds designed to limit mutagenesis in a pathogen increasingly associated with drug resistance.