Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of <i>Mycobacterium smegmatis</i> Polynucleotide Phosphorylase

Unciuleac, Mihaela-Carmen; Shuman, Stewart

doi:10.1021/bi400281w

Cited by 11 publications

(41 citation statements)

References 37 publications

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“…Our findings on the ssDNA polymerase and phosphorylase catalytic activities of Rv2783 contribute to an emerging picture of mycobacterial PNPases as catalysts of DNA metabolism, in addition to their synthetic and degradative roles in RNA metabolism (16). As suggested, Rv2783 is likely involved in conserving DNA integrity through DNA repair and mutagenesis in vivo.…”

Section: Discussionmentioning

confidence: 57%

“…Rv2783 is predicted to function as a PNPase enzyme, which catalyzes synthetic and degradative activities in RNA metabolism (15). Exemplary bacterial PNPase enzymes have been reported to catalyze DNA activities in vitro when manganese or iron is provided in lieu of magnesium as the metal cofactor (16). Therefore, it was of considerable relevance to determine whether Rv2783 could catalyze single-stranded DNA (ssDNA) polymerization and phosphorolysis activities.…”

Section: Resultsmentioning

confidence: 99%

“…The DNA/RNA polymerization and phosphorolytic activities of Rv2783 were determined according to previously published work on M. smegmatis PNPase (16), which served as a positive control, with some modifications. ssDNA polymerization assay.…”

Section: Methodsmentioning

confidence: 99%

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Pyrazinoic Acid Inhibits a Bifunctional Enzyme in Mycobacterium tuberculosis

Njire

Wang

et al. 2017

Antimicrob Agents Chemother

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Pyrazinamide (PZA), an indispensable component of modern tuberculosis treatment, acts as a key sterilizing drug. While the mechanism of activation of this prodrug into pyrazinoic acid (POA) by Mycobacterium tuberculosis has been extensively studied, not all molecular determinants that confer resistance to this mysterious drug have been identified. Here, we report how a new PZA resistance determinant, the Asp67Asn substitution in Rv2783, confers M. tuberculosis resistance to PZA. Expression of the mutant allele but not the wild-type allele in M. tuberculosis recapitulates the PZA resistance observed in clinical isolates. In addition to catalyzing the metabolism of RNA and single-stranded DNA, Rv2783 also metabolized ppGpp, an important signal transducer involved in the stringent response in bacteria. All catalytic activities of the wild-type Rv2783 but not the mutant were significantly inhibited by POA. These results, which indicate that Rv2783 is a target of PZA, provide new insight into the molecular mechanism of the sterilizing activity of this drug and a basis for improving the molecular diagnosis of PZA resistance and developing evolved PZA derivatives to enhance its antituberculosis activity.

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Section: Discussionmentioning

confidence: 57%

Section: Resultsmentioning

confidence: 99%

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Pyrazinoic Acid Inhibits a Bifunctional Enzyme in Mycobacterium tuberculosis

Njire

Wang

et al. 2017

Antimicrob Agents Chemother

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“…E. coli PNPase comprises part of a larger macromolecular RNA processing machine, the RNA degradosome, that includes an RNA helicase, RhlB (34,35). Recombinant mycobacterial PNPase has been purified and characterized biochemically (36,37), but its potential interaction partners and genetic dissection of its physiology are uncharted territory. Indeed, little is known about RNA turnover in mycobacteria.…”

Section: Discussionmentioning

confidence: 99%

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids

2015

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Mycobacteria have a large and distinctive ensemble of DNA helicases that function in DNA replication, repair, and recombination. Little is known about the roster of RNA helicases in mycobacteria or their roles in RNA transactions. The 912-amino-acid Mycobacterium smegmatis HelY (MSMEG_3885) protein is a bacterial homolog of the Mtr4 and Ski2 helicases that regulate RNA 3= processing and turnover by the eukaryal exosome. Here we characterize HelY as an RNA-stimulated ATPase/dATPase and an ATP/dATP-dependent 3=-to-5= helicase. HelY requires a 3= single-strand RNA tail (a loading RNA strand) to displace the complementary strand of a tailed RNA:RNA or RNA:DNA duplex. The findings that HelY ATPase is unresponsive to a DNA polynucleotide cofactor and that HelY is unable to unwind a 3=-tailed duplex in which the loading strand is DNA distinguish HelY from other mycobacterial nucleoside triphosphatases/helicases characterized previously. The biochemical properties of HelY, which resemble those of Mtr4/Ski2, hint at a role for HelY in mycobacterial RNA catabolism. H elicases are nucleic acid-dependent nucleoside triphosphatases (NTPases) that play important roles in diverse nucleic acid transactions. DNA helicases orchestrate replication, recombination, and repair. RNA helicases choreograph transcription, RNA processing, ribosome biogenesis, translation, and RNA turnover. Helicases use the chemical energy of nucleoside triphosphate (NTP) hydrolysis either to effect mechanical changes in the secondary structure of nucleic acids or to remodel (or disrupt) the structures of protein-nucleic acid complexes. Helicases are classified into superfamilies, families, and subfamilies according to their distinctive primary, tertiary, and quaternary structures and their biochemical specificities, i.e., NTP preference, nucleic acid preference (duplex DNA, duplex RNA, or RNA:DNA hybrids), and directionality of translocation or unwinding (5= to 3= or 3= to 5=) (1).Differences in the roster of helicases between taxa offer useful clues to the evolution and diversification of DNA replication/repair and RNA synthesis/processing strategies. Where the helicase rosters diverge in animals versus pathogens, they can suggest antiinfective drug targets. With this in mind, we are focused on the helicases of members of the genus Mycobacterium, a genus of the phylum Actinobacteria that includes the human pathogen Mycobacterium tuberculosis and its avirulent relative, M. smegmatis. To date, we have purified and characterized six mycobacterial DNA helicases: AdnAB (2, 3, 4), UvrD1 (5, 6), UvrD2 (7), SftH (8), RqlH (9), and Lhr (10). Mycobacterial DNA helicases XBP (11, 12), RecG (13,14,15), RecD (16), RuvB (17), and DnaB (18) have been purified and characterized by other investigators.Comparatively little attention has been paid to the mycobacterial RNA helicase repertoire. The transcription termination factor Rho is, to our knowledge, the lone example of a biochemically characterized NTP-driven RNA translocase/helicase from a Mycobacterium spec...

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“…Remarkably, PNPase can catalyse both DNA phosphorolysis and template independent synthesis of DNA from dNDPs [9–15]. The latter enzymatic property was exploited in the early era of molecular biology for the synthesis of oligodeoxyribonucleotides but was not generally considered to play a role in vivo.…”

Section: Introductionmentioning

confidence: 99%

Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli

et al. 2017

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BackgroundPolynucleotide phosphorylase (PNPase, encoded by pnp) is generally thought of as an enzyme dedicated to RNA metabolism. The pleiotropic effects of PNPase deficiency is imputed to altered processing and turnover of mRNAs and small RNAs, which in turn leads to aberrant gene expression. However, it has long since been known that this enzyme may also catalyze template-independent polymerization of dNDPs into ssDNA and the reverse phosphorolytic reaction. Recently, PNPase has been implicated in DNA recombination, repair, mutagenesis and resistance to genotoxic agents in diverse bacterial species, raising the possibility that PNPase may directly, rather than through control of gene expression, participate in these processes.ResultsIn this work we present evidence that in Escherichia coli PNPase enhances both homologous recombination upon P1 transduction and error prone DNA repair of double strand breaks induced by zeocin, a radiomimetic agent. Homologous recombination does not require PNPase phosphorolytic activity and is modulated by its RNA binding domains whereas error prone DNA repair of zeocin-induced DNA damage is dependent on PNPase catalytic activity and cannot be suppressed by overexpression of RNase II, the other major enzyme (encoded by rnb) implicated in exonucleolytic RNA degradation. Moreover, E. coli pnp mutants are more sensitive than the wild type to zeocin. This phenotype depends on PNPase phosphorolytic activity and is suppressed by rnb, thus suggesting that zeocin detoxification may largely depend on RNA turnover.ConclusionsOur data suggest that PNPase may participate both directly and indirectly through regulation of gene expression to several aspects of DNA metabolism such as recombination, DNA repair and resistance to genotoxic agents.

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Distinctive Effects of Domain Deletions on the Manganese-Dependent DNA Polymerase and DNA Phosphorylase Activities of Mycobacterium smegmatis Polynucleotide Phosphorylase

Cited by 11 publications

References 37 publications

Pyrazinoic Acid Inhibits a Bifunctional Enzyme in Mycobacterium tuberculosis

Pyrazinoic Acid Inhibits a Bifunctional Enzyme in Mycobacterium tuberculosis

Mycobacterium smegmatis HelY Is an RNA-Activated ATPase/dATPase and 3′-to-5′ Helicase That Unwinds 3′-Tailed RNA Duplexes and RNA:DNA Hybrids

Polynucleotide phosphorylase is implicated in homologous recombination and DNA repair in Escherichia coli

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