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Tobacco-specific nitrosamine-derived O2-alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli.

ABSTRACT: To investigate the biological effects of the O(2)-alkylthymidines induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), we have replicated a plasmid containing O(2)-methylthymidine (O(2)-Me-dT) or O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine (O(2)-POB-dT) in Escherichia coli with specific DNA polymerase knockouts. High genotoxicity of the adducts was manifested in the low yield of transformants from the constructs, which was 2-5% in most strains but increased 2-4-fold with SOS. In the SOS-induced wild type E. coli, O(2)-Me-dT and O(2)-POB-dT induced 21% and 56% mutations, respectively. For O(2)-POB-dT, the major type of mutation was T ? G followed by T ? A, whereas for O(2)-Me-dT, T ? G and T ? A occurred in equal frequency. For both lesions, T ? C also was detected in low frequency. The T ? G mutation was reduced in strains with deficiency in any of the three SOS polymerases. By contrast, T ? A was abolished in the pol V(-) strain, while its frequency in other strains remained unaltered. This suggests that pol V was responsible for the T ? A mutations. The potent mutagenicity of these lesions may be related to NNK mutagenesis and carcinogenesis.

SUBMITTER: Jasti VP

PROVIDER: S-EPMC3221470 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Tobacco-specific nitrosamine-derived O2-alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli.

Jasti Vijay P VP Spratt Thomas E TE Basu Ashis K AK

Chemical research in toxicology 20111028 11

To investigate the biological effects of the O(2)-alkylthymidines induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), we have replicated a plasmid containing O(2)-methylthymidine (O(2)-Me-dT) or O(2)-[4-(3-pyridyl-4-oxobut-1-yl]thymidine (O(2)-POB-dT) in Escherichia coli with specific DNA polymerase knockouts. High genotoxicity of the adducts was manifested in the low yield of transformants from the constructs, which was 2-5% in most strains but incr ...[more]

PMID: 22029400

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Project description:BackgroundDNA damage in Escherichia coli evokes a response mechanism called the SOS response. The genetic circuit of this mechanism includes the genes recA and lexA, which regulate each other via a mixed feedback loop involving transcriptional regulation and protein-protein interaction. Under normal conditions, recA is transcriptionally repressed by LexA, which also functions as an auto-repressor. In presence of DNA damage, RecA proteins recognize stalled replication forks and participate in the DNA repair process. Under these conditions, RecA marks LexA for fast degradation. Generally, such mixed feedback loops are known to exhibit either bi-stability or a single steady state. However, when the dynamics of the SOS system following DNA damage was recently studied in single cells, ordered peaks were observed in the promoter activity of both genes (Friedman et al., 2005, PLoS Biol. 3(7):e238). This surprising phenomenon was masked in previous studies of cell populations. Previous attempts to explain these results harnessed additional genes to the system and deployed complex deterministic mathematical models that were only partially successful in explaining the results.Methodology/principal findingsHere we apply stochastic methods, which are better suited for dynamic simulations of single cells. We show that a simple model, involving only the basic components of the circuit, is sufficient to explain the peaks in the promoter activities of recA and lexA. Notably, deterministic simulations of the same model do not produce peaks in the promoter activities.Conclusion/significanceWe conclude that the double negative mixed feedback loop with auto-repression accounts for the experimentally observed peaks in the promoter activities. In addition to explaining the experimental results, this result shows that including additional regulations in a mixed feedback loop may dramatically change the dynamic functionality of this regulatory module. Furthermore, our results suggests that stochastic fluctuations strongly affect the qualitative behavior of important regulatory modules even under biologically relevant conditions, thus emphasizing the importance of stochastic analysis of regulatory circuits.

Project description:In bacteria, SOS is a global response to DNA damage, mediated by the recA-lexA genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that Escherichia coli responds to DNA damage via another recA-lexA-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called extensive-damage-induced (Edin) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH? through the Fenton reaction, eventually resulting in cell death. Our genetic and molecular studies on ALD provide additional insight for the evolution of mitochondria and the apoptotic pathway in eukaryotes. Importance: The SOS response is the first described and the most studied bacterial response to DNA damage. It is mediated by a set of two genes, recA-lexA, and it results in DNA repair and thereby in the survival of the bacterial culture. We have shown that Escherichia coli responds to DNA damage by an additional recA-lexA-mediated pathway resulting in an apoptosis-like death (ALD). Apoptosis is a mode of cell death that has previously been reported only in eukaryotes. We found that E. coli ALD is characterized by several hallmarks of eukaryotic mitochondrial apoptosis. Altogether, our results revealed that recA-lexA is a DNA damage response coordinator that permits two opposite responses: life, mediated by the SOS, and death, mediated by the ALD. The choice seems to be a function of the degree of DNA damage in the cell.

Dataset Information

Tobacco-specific nitrosamine-derived O2-alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli.

Publications

Tobacco-specific nitrosamine-derived O2-alkylthymidines are potent mutagenic lesions in SOS-induced Escherichia coli.

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