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

ABSTRACT: Polynucleotide 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.In 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.Our 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.

SUBMITTER: Carzaniga T

PROVIDER: S-EPMC5379764 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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

Carzaniga Thomas T Sbarufatti Giulia G Briani Federica F Dehò Gianni G

BMC microbiology 20170404 1

<h4>Background</h4>Polynucleotide 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 ...[more]

PMID: 28376742

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Project description:Despite the identification of many genes and pathways involved in the persistence phenomenon in bacteria, the mechanisms of persistence are not well understood. Here, using Escherichia coli, we identified polynucleotide phosphorylase (PNPase) as a key regulator of persister formation. We constructed the pnp knockout strain (Δpnp) and its complemented strain and exposed them to antibiotics and stress conditions. The results showed that, compared with the wild-type strain W3110, the Δpnp strain had significant defects in persistence to antibiotics and stresses, and the persistence phenotype was restored upon complementation with the pnp gene. Transcriptome sequencing (RNA-seq) analysis revealed that 242 (166 upregulated and 76 downregulated) genes were differentially expressed in the Δpnp strain compared with the W3110 strain. KEGG analysis of the upregulated genes showed that these genes were mostly mapped to metabolism and virulence pathways, of which most are positively regulated by the global regulator cyclic AMP receptor protein (CRP). Correspondingly, the transcription level of the crp gene in the Δpnp strain increased 3.22-fold in the early stationary phase. We further explored the indicators of cellular metabolism of the Δpnp strain, the phenotype of the pnp and crp double-deletion mutant, and the transcriptional activity of the crp gene. Our results indicate that PNPase controls cellular metabolism by negatively regulating the crp operon via targeting the 5'-untranslated region of the crp transcript. This study reveals a persister mechanism and provides novel targets for the development of drugs against persisters for more effective treatment. IMPORTANCE Persisters pose significant challenges for a more effective treatment of persistent infections. An improved understanding of mechanisms of persistence will provide therapeutic targets important for the development of better treatments. Since recent studies with the key tuberculosis persister drug pyrazinamide have implicated polynucleotide phosphorylase (PNPase) as a drug target, in this study, we addressed the possibility that PNPase might be involved in persistence in Escherichia coli. Our study demonstrates PNPase indeed being involved in persistence, provides a mechanism by which PNPase controls persister formation, and suggests a new therapeutic target for treating persistent bacterial infections.

Project description:UnlabelledThe complex posttranscriptional regulation mechanism of the Escherichia coli pnp gene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed for pnp autoregulation posit that the target of PNPase is a mature pnp mRNA previously processed at its 5' end by RNase III, rather than the primary pnp transcript (RNase III-dependent models), and that PNPase activity eventually leads to pnp mRNA degradation by RNase E. However, some published data suggest that pnp expression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δpnp rnc(+) and Δpnp Δrnc strains with a chromosomal pnp-lacZ translational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition for pnp primary transcript between PNPase and the ribosomal protein S1.ImportanceIn Escherichia coli, polynucleotide phosphorylase (PNPase, encoded by pnp) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of the pnp primary transcript, creates the substrate for PNPase regulatory activity, eventually leading to pnp mRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism of pnp mRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability.

Dataset Information

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

Publications

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

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