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

In Escherichia coli themutT 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 vitrothe 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 mutTmutants 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 μm. When 8-oxo-dGTP was added at 0.34 μm 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.

• Publication year

  2002

• Venue

  Journal of Biological Chemistry

• Publication date

  2002-05-03

• Fields of study

  Biology, Medicine, Chemistry

• Identifiers
  DOI 10.1074/JBC.M200965200PMID 11856756
• External record

  Open on Semantic Scholar

• Source metadata

  Semantic Scholar, PubMed

• No claims are published for this paper.

• No concepts are published for this paper.

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