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Transcription increases methylmethane sulfonate-induced mutations in alkB strains of Escherichia coli.

ABSTRACT: Methylmethane sulfonate (MMS) produces DNA base lesions, including 3-methylcytosine (m3C), more effectively in single-stranded DNA. The repair of m3C in Escherichia coli is mediated by AlkB through oxidative demethylation and in the absence of repair, m3C leads to base-substitution mutations. We describe here results of experiments that were designed to investigate whether transcription of a gene in E. coli affects the process of mutagenesis by MMS and the roles played by AlkB and lesion bypass polymerase PolV. Using a genetic reversion assay, we have confirmed that MMS mutagenesis is suppressed by AlkB, but is enhanced by PolV. High transcription of the target gene enhances reversion frequency in an orientation-dependent manner. When the cytosines that are the likely targets of MMS were in the non-template strand (NTS), transcription increased the MMS-induced reversion frequency several fold. This increase was dependent on the presence of PolV. In contrast, when the same cytosines were present in the template strand, transcription had little effect on reversion frequency induced by MMS. These data suggest that MMS creates 3-methylcytosine adducts in the NTS and are consistent with an idea proposed previously that transcription makes the NTS transiently single-stranded and more accessible to chemicals. We propose that this is the underlying cause of its increased sensitivity to MMS and suggest that transcriptionally active DNA may be a preferred target for the action of alkylating agents that prefer single-stranded DNA.

SUBMITTER: Fix D

PROVIDER: S-EPMC2569841 | biostudies-literature | 2008 Aug

REPOSITORIES: biostudies-literature

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Transcription increases methylmethane sulfonate-induced mutations in alkB strains of Escherichia coli.

Fix Douglas D Canugovi Chandrika C Bhagwat Ashok S AS

DNA repair 20080602 8

Methylmethane sulfonate (MMS) produces DNA base lesions, including 3-methylcytosine (m3C), more effectively in single-stranded DNA. The repair of m3C in Escherichia coli is mediated by AlkB through oxidative demethylation and in the absence of repair, m3C leads to base-substitution mutations. We describe here results of experiments that were designed to investigate whether transcription of a gene in E. coli affects the process of mutagenesis by MMS and the roles played by AlkB and lesion bypass ...[more]

PMID: 18515192

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Project description:BackgroundThe rational design of L-phenylalanine (L-Phe) overproducing microorganisms has been successfully achieved by combining different genetic strategies such as inactivation of the phosphoenolpyruvate: phosphotransferase transport system (PTS) and overexpression of key genes (DAHP synthase, transketolase and chorismate mutase-prephenate dehydratase), reaching yields of 0.33 (g-Phe/g-Glc), which correspond to 60% of theoretical maximum. Although genetic modifications introduced into the cell for the generation of overproducing organisms are specifically targeted to a particular pathway, these can trigger unexpected transcriptional responses of several genes. In the current work, metabolic transcription analysis (MTA) of both L-Phe overproducing and non-engineered strains using Real-Time PCR was performed, allowing the detection of transcriptional responses to PTS deletion and plasmid presence of genes related to central carbon metabolism. This MTA included 86 genes encoding enzymes of glycolysis, gluconeogenesis, pentoses phosphate, tricarboxylic acid cycle, fermentative and aromatic amino acid pathways. In addition, 30 genes encoding regulatory proteins and transporters for aromatic compounds and carbohydrates were also analyzed.ResultsMTA revealed that a set of genes encoding carbohydrate transporters (galP, mglB), gluconeogenic (ppsA, pckA) and fermentative enzymes (ldhA) were significantly induced, while some others were down-regulated such as ppc, pflB, pta and ackA, as a consequence of PTS inactivation. One of the most relevant findings was the coordinated up-regulation of several genes that are exclusively gluconeogenic (fbp, ppsA, pckA, maeB, sfcA, and glyoxylate shunt) in the best PTS- L-Phe overproducing strain (PB12-ev2). Furthermore, it was noticeable that most of the TCA genes showed a strong up-regulation in the presence of multicopy plasmids by an unknown mechanism. A group of genes exhibited transcriptional responses to both PTS inactivation and the presence of plasmids. For instance, acs-ackA, sucABCD, and sdhABCD operons were up-regulated in PB12 (PTS mutant that carries an arcB- mutation). The induction of these operons was further increased by the presence of plasmids in PB12-ev2. Some genes involved in the shikimate and specific aromatic amino acid pathways showed down-regulation in the L-Phe overproducing strains, might cause possible metabolic limitations in the shikimate pathway.ConclusionThe identification of potential rate-limiting steps and the detection of transcriptional responses in overproducing microorganisms may suggest "reverse engineering" strategies for the further improvement of L-Phe production strains.

Project description:The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In Escherichia coli, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that E. coli NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (Δndk) in E. coli ΔmutT or ΔmutT ΔribA strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in E. coliIMPORTANCE Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in Escherichia coli The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.

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Transcription increases methylmethane sulfonate-induced mutations in alkB strains of Escherichia coli.

Publications

Transcription increases methylmethane sulfonate-induced mutations in alkB strains of Escherichia coli.

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