Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis.
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 beta-clamp. Moreover, disruption of the ImuB-beta 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 alpha-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.
Warner
Digby F
DF
Medical Research Council/National Health Laboratory Service/University of the Witwatersrand Molecular Mycobacteriology Research Unit and Department of Science and Technology/National Research Foundation Centre of Excellence for Biomedical Tuberculosis Research, Faculty of Health Sciences, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg 2000, South Africa. digby.warner@nhls.ac.za
Ndwandwe
Duduzile E
DE
Abrahams
Garth L
GL
Kana
Bavesh D
BD
Machowski
Edith E
EE
Venclovas
Ceslovas
C
Mizrahi
Valerie
V
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D055162:Biocatalysis; D020134:Catalytic Domain; D004249:DNA Damage; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011485:Protein Binding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004259:DNA-Directed DNA Polymerase