Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

MRC/NHLS/WITS Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, School of Pathology of the University of the Witwatersrand and the National Health Laboratory Service, Johannesburg, South Africa.
Journal of bacteriology (Impact Factor: 2.81). 02/2010; 192(8):2220-7. DOI: 10.1128/JB.01135-09
Source: PubMed


The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion
synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however,
their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical
C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N2-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate
of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred
a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.

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Available from: Neil G Stoker, Aug 25, 2014
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    • "These studies implied that DNAE2 is the primary enzyme responsible for adaptive mutagenesis in case of Mtb [14]. Additionally, genetic studies probing for the effect of loss of function of DinB homologs in Mtb showed no significant changes in the phenotype [15]. This study also showed that there was no increase in the frequency of mutations when these homologs were expressed ectopically in Msm, and the authors suggest that mycobacterial DinB homologs function differently from those in other bacteria. "
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    ABSTRACT: Error-prone DNA synthesis in prokaryotes imparts plasticity to the genome to allow for evolution in unfavorable environmental conditions, and this phenomenon is termed adaptive mutagenesis. At a molecular level, adaptive mutagenesis is mediated by upregulating the expression of specialized error-prone DNA polymerases that generally belong to the Y-family, such as the polypeptide product of the dinB gene in case of E. coli. However, unlike E. coli, it has been seen that expression of the homologs of dinB in Mycobacterium tuberculosis are not upregulated under conditions of stress. These studies suggest that DinB homologs in Mycobacteria might not be able to promote mismatches and participate in adaptive mutagenesis. We show that a representative homolog from Mycobacterium smegmatis (MsDpo4) can carry out template-dependent nucleotide incorporation and therefore is a DNA polymerase. In addition, it is seen that MsDpo4 is also capable of misincorporation with a significant ability to promote G:T and T:G mismatches. The frequency of misincorporation for these two mismatches is similar to that exhibited by archaeal and prokaryotic homologs. Overall, our data show that MsDpo4 has the capacity to facilitate transition mutations and can potentially impart plasticity to the genome.
    Full-text · Article · Mar 2012 · Journal of nucleic acids
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    • "dinB-encoded Pol IV catalyzes error-prone translesion synthesis (TLS) in Escherichia coli and several bacteria. However, in M. tuberculosis, deletion of two dinB homologs individually or in combination had no effect on the susceptibility to compounds that form N2-dG adducts and alkylating agents, and the rate and the spectrum of spontaneous mutations (14). It was suggested that the DinB homologs in Mycobacterium differ in biological functions from their counterparts in other bacteria. "
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    ABSTRACT: Linear chromosomes and linear plasmids of Streptomyces are capped by terminal proteins that are covalently bound to the 5′-ends of DNA. Replication is initiated from an internal origin, which leaves single-stranded gaps at the 3′-ends. These gaps are patched by terminal protein-primed DNA synthesis. Streptomyces contain five DNA polymerases: one DNA polymerase I (Pol I), two DNA polymerases III (Pol III) and two DNA polymerases IV (Pol IV). Of these, one Pol III, DnaE1, is essential for replication, and Pol I is not required for end patching. In this study, we found the two Pol IVs (DinB1 and DinB2) to be involved in end patching. dinB1 and dinB2 could not be co-deleted from wild-type strains containing a linear chromosome, but could be co-deleted from mutant strains containing a circular chromosome. The resulting ΔdinB1 ΔdinB2 mutants supported replication of circular but not linear plasmids, and exhibited increased ultraviolet sensitivity and ultraviolet-induced mutagenesis. In contrast, the second Pol III, DnaE2, was not required for replication, end patching, or ultraviolet resistance and mutagenesis. All five polymerase genes are relatively syntenous in the Streptomyces chromosomes, including a 4-bp overlap between dnaE2 and dinB2. Phylogenetic analysis showed that the dinB1-dinB2 duplication occurred in a common actinobacterial ancestor.
    Full-text · Article · Feb 2012 · Nucleic Acids Research
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    • "The results presented by Gupta et al. are consistent with the idea that mycobacterial repair pathways might differ fundamentally from the standard models. Other examples include the involvement of non-orthologous analogues of DNA repair components in core systems (Boshoff et al., 2003; Warner et al., 2010) as well as the presence of multiple homologues of some repair genes with cryptic (Kana et al., 2010) or specialized (Guo et al., 2010) functions. In combination, these and related studies suggest that that existing models of bacterial DNA replication and repair should be expanded to include M. tuberculosis. "
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    ABSTRACT: Genotoxic agents from endogenous and exogenous sources cause double-strand breaks (DSBs) in chromosomal DNA. Given the threat these lesions pose to viability, it is not surprising that multiple, conserved mechanisms exist for their detection, processing and repair. Previous studies have established both functional non-homologous end-joining (NHEJ) and homologous recombination (HR) systems in mycobacteria. However, relative pathway utilization in these organisms, which include the major human pathogen Mycobacterium tuberculosis, remains unclear. In this issue, Glickman and colleagues describe an elegant assay to distinguish DSB repair outcomes through simple phenotypic screening. By applying their novel reporter system to a panel of repair pathway mutants, they identify an unexpected role for single-strand annealing (SSA) in the related non-pathogen, Mycobacterium smegmatis. As such, these results expand the mycobacterial DSB repair pathway complement to three mechanisms that are distinguishable by their differential requirements for the DSB-resecting, helicase-nuclease machines, AdnAB and RecBCD. Notably, in an unexpected departure from classical models, they establish that mycobacterial RecBCD is a dedicated SSA nuclease, while AdnAB is required for RecA-dependent HR. Here, we consider the implications of their observations, which include the asymmetric cross-regulation of pathway function, for the role of DSB repair in mycobacterial pathogenesis.
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