Essential roles for imuA'- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis.
ABSTRACT 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.
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ABSTRACT: Mycobacterium smegmatis DinB2 is the founder of a clade of Y-family DNA polymerase that is naturally adept at utilizing rNTPs or dNTPs as substrates. Here we investigate the fidelity and lesion bypass capacity of DinB2. We report that DinB2 is an unfaithful DNA and RNA polymerase with a distinctive signature for misincorporation of dNMPs, rNMPs and oxoguanine nucleotides during templated synthesis in vitro. DinB2 has a broader mutagenic spectrum with manganese than magnesium, though low ratios of manganese to magnesium suffice to switch DinB2 to its more mutagenic mode. DinB2 discrimination against incorrect dNTPs in magnesium is primarily at the level of substrate binding affinity, rather than kpol. DinB2 can incorporate any dNMP or rNMP opposite oxo-dG in the template strand with manganese as cofactor, with a kinetic preference for synthesis of an A:oxo-dG Hoogsteen pair. With magnesium, DinB2 is adept at synthesizing A:oxo-dG or C:oxo-dG pairs. DinB2 effectively incorporates deoxyribonucleotides, but not ribonucleotides, opposite an abasic site, with kinetic preference for dATP as the substrate. We speculate that DinB2 might contribute to mycobacterial mutagenesis, oxidative stress and quiescence, and discuss the genetic challenges to linking the polymerase biochemistry to an in vivo phenotype.Nucleic Acids Research 10/2014; 42(20). DOI:10.1093/nar/gku1027 · 8.81 Impact Factor
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ABSTRACT: New drugs that retain potency against multidrug/extensively drug-resistant strains of Mycobacterium tuberculosis, with the additional benefit of a shortened treatment duration and ease of administration, are urgently needed by tuberculosis (TB) control programs. Efforts to develop this new generation of treatment interventions have been plagued with numerous problems, the most significant being our insufficient understanding of mycobacterial metabolism during disease. This, combined with limited chemical diversity and poor entry of small molecules into the cell, has limited the number of new bioactive agents that result from drug screening efforts. The biochemical, target-driven approach to drug development has been largely abandoned in the TB field, to be replaced by whole-cell or target-based whole-cell screening approaches. In this context, the properties of a good drug target are unclear, since these are directly determined by the ability to find compounds, using current screening algorithms, which are able to kill M. tuberculosis. In this review, we discuss issues related to the identification and validation of drug targets and highlight some key properties for promising targets. Some of these include essentiality for growth, vulnerability, druggability, reduced propensity to evolve drug resistance and target location to facilitate ready access to drugs during chemotherapy. We present these in the context of recent drugs that have emerged through various approaches with the aim of consolidating the knowledge gained from these experiences to inform future efforts.Tuberculosis 10/2014; 94(6). DOI:10.1016/j.tube.2014.10.003 · 3.50 Impact Factor
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ABSTRACT: Different methods have been developed to infer regulatory networks from heterogeneous omics datasets and to construct co-expression networks. Each algorithm produces different networks and efforts have been devoted to automatically integrate them into consensus sets. However each separate set has an intrinsic value that is diluted and partly lost when building a consensus network. Here we present a methodology to generate co-expression networks and, instead of a consensus network, we propose an integration framework where the different networks are kept and analysed with additional tools to efficiently combine the information extracted from each network.BMC Systems Biology 09/2014; 8(1):111. DOI:10.1186/s12918-014-0111-5 · 2.85 Impact Factor