Assessing the Metabolic Function of the MutT 8-Oxodeoxyguanosine Triphosphatase in Escherichia coli by Nucleotide Pool Analysis

Mary Lynn Tassotto,Christopher K Mathews

doi:10.1074/jbc.m200965200

Abstract

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-oxo-dGTP 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-oxo-dGTP content of these bacteria is about 200 molecules/cell, corresponding to a concentration of about 0.34 microm. When 8-oxo-dGTP was added at 0.34 microm 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.

Highlights

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]
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 Km 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
We have developed a separation and analytical system that allows the detection of as little as 6 pmol of 8-oxo-dGTP in a bacterial extract containing the normal nucleotides at their ordinary concentrations

Summary

EXPERIMENTAL PROCEDURES

Mutant E. coli Strains and Culture Conditions—E. coli strains used included strain B, the wild-type control from our collection, and three mutT mutants obtained from the E. coli Genetic Stock Center, New Haven, CT. Injections (50 ␮l each) of bacterial extract were performed in duplicate with material in the second injection containing internal standards of dATP, dCTP, dGTP, dTTP, and 8-oxodGTP Data from these second runs were used to verify identifications made from elution times, as well as to calculate recoveries of each nucleotide and apply these figures to correct nucleotide data from the extract for variable losses of material during nucleotide extraction and separation. The reaction mixture was analyzed by HPLC using an Alltima C18 column (5 ␮m, 250 ϫ 4.6 mm, Alltech Inc.) at a flow rate of 1.0 ml/min, with nucleotides detected by UV absorbance at 293 nm. DNA synthesis reactions were carried out using HeLa S3 cell cytoplasmic extract (75 ␮g protein), 1 ␮g of SV40 T antigen (Molecular Biology Resources, Milwaukee, WI), 80 ng of DNA template, and nucleotides at the following concentrations: dATP, 60 ␮M; dCTP, 30 ␮M; dGTP, 10 ␮M; dTTP, 60 ␮M; and 8-oxo-dGTP (Amersham Biosciences); concentrations varied as indicated.

RESULTS

DISCUSSION

Background