In Escherichia coli the mutT gene is one of several that acts to minimize mutagenesis by reactive oxygen species. The bacterial MutT protein and its mammalian homolog have been shown to catalyze in vitro the hydrolysis of the oxidized deoxyguanosine nucleotide, 8-oxo-dGTP, to its corresponding monophosphate. Thus, the protein is thought to "sanitize" the nucleotide pool by ridding the cell of a nucleotide whose incorporation into DNA would be intensely mutagenic. However, because others have shown mutT mutations to be mutagenic under some conditions of anaerobic growth, and have shown 8-oxo-dGTP to be a poor DNA polymerase substrate, there is reason to question this model. We have devised an assay for 8-oxo-dGTP in bacterial extracts. Using this assay, which involves reversed-phase high-performance liquid chromatography and electrochemical detection, we have been unable to detect 8-oxodGTP in extracts of three different mutT mutants of E. coli, even after growth of the bacteria in the presence of hydrogen peroxide. Our estimated upper limit for 8-oxodGTP content of these bacteria is about 200 molecules/ cell, corresponding to a concentration of about 0.34 M. When 8-oxo-dGTP was added at 0.34 M to an in vitro DNA replication system primed with a DNA template that permits scoring of replication errors and with the four normal dNTPs at their estimated intracellular concentrations, there was no detectable effect upon the frequency of replication errors. These findings lead us to question the conclusion that 8-oxo-dGTP is the most significant physiological substrate for the MutT protein.The action of reactive oxygen species upon cells stimulates mutagenesis in large part by increasing the abundance in DNA of the oxidized guanine derivative, 7,8-dihydro-8-oxoguanine (8-oxoG).1 This base efficiently pairs with adenine, leading, if unrepaired, to a transversion mutation (1). In Escherichia coli the products of three genes play particularly prominent roles in counteracting this genotoxicity, and all three have homologs in mammalian cells. Two of these genes, mutM and mutY, encode DNA glycosylases, and their actions initiate base excision repair processes at sites occupied by oxoG (1). The third gene, mutT, encodes a nucleotidase, which cleaves dGTP to dGMP and pyrophosphate (2) but which Maki and Sekiguchi (3) showed to have much lower K m for the oxidized dGTP derivative, 8-oxo-dGTP. This finding plus subsequent publications from the same laboratory (4 -9) support the concept that the action of the MutT protein is to "sanitize" the nucleotide pool by removing from cells a damaged nucleotide that, if incorporated into DNA, would be strongly mutagenic. In agreement with this model, several investigators (10 -12) have shown that addition of 8-oxo-dGTP to an in vitro DNA replication system in which replication errors could be scored as mutations stimulated replication errors that were shown by sequence analysis to be transversions.The human homolog of MutT, hMTH1, has been expressed in mutT mutant E. coli and shown to su...
While investigating the basis for marked natural asymmetries in deoxyribonucleoside triphosphate (dNTP) pools in mammalian cells, we observed that culturing V79 hamster lung cells in a 2% oxygen atmosphere causes 2-3-fold expansions of the dATP, dGTP, and dTTP pools, whereas dCTP declines by a comparable amount. Others have made similar observations and have proposed that, because O 2 is required for formation of the catalytically essential oxygen-bridged iron center in ribonucleotide reductase, dCTP depletion at low oxygen tension results from direct or indirect effects upon ribonucleotide reductase. We have tested the hypothesis that oxygen limitation affects ribonucleotide specificity using recombinant mouse ribonucleotide reductase and an assay that permits simultaneous monitoring of the reduction of all four nucleotide substrates. Preincubation and assay of the enzyme in an anaerobic chamber caused only partial activity loss. Accordingly, we treated the enzyme with hydroxyurea, followed by removal of the hydroxyurea and exposure to atmospheres of varying oxygen content. The activity was totally depleted by hydroxyurea treatment and nearly fully regained by exposure to air. By the criterion of activities regained at different oxygen tensions, we found CDP reduction not to be specifically sensitive to oxygen depletion; however, GDP reduction was specifically sensitive. The basis for the differential response to reactivation by O 2 is not known, but it evidently does not involve varying rates of reactivation of different allosteric forms of the enzyme or altered response to allosteric effectors at reduced oxygen tension.This investigation arose from our attempts to identify the biochemical basis for a natural asymmetry in the pool sizes of the four deoxyribonucleoside triphosphates (dNTPs) in mammalian cells (1). In nearly every cell line analyzed, dGTP represents just 5-10% of the sum of the four dNTP pools (dATP ϩ dCTP ϩ dGTP ϩ dTTP).A possible explanation for this asymmetry came from the discovery that the mutT gene product in Escherichia coli is a nucleoside triphosphatase, with particular activity against an oxidized guanine nucleotide, 8-oxo-dGTP (2), and that mammalian cells contain a similar enzyme (3). An 8-oxo-dGMP residue in DNA stimulates mutagenesis via a transversion pathway because of its ready tendency to base pair with dAMP (4). Therefore, the mutT gene product and its homolog are thought to minimize replication errors by eliminating from the cell an oxidized nucleotide, 8-oxo-dGTP, whose incorporation into DNA would otherwise be a strongly mutagenic event.We hypothesized that turnover of dGTP, via its oxidation and subsequent degradation, might contribute to its underrepresentation in the DNA precursor pool, and we asked whether culturing cells at low oxygen tension, where guanine nucleotide oxidation might be minimized, would cause dGTP to accumulate. However, as detailed below, we found that culturing V79 hamster lung cells in an artificial atmosphere containing 2% oxygen instead of at its ...
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