Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool.

Abstract

8-0xguanie nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies.Once 8-oxodGTP is formed in the cellular nucleotide po-, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein ofEscherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified >400-fold.Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a K.m value of 12.5 pM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.Mutator mutants that show an increased frequency of spontaneous mutations have led to elucidation of the multiple pathways of spontaneous mutagenesis. Studies on Escherichia coli mutator genes and their products revealed that a major cause of spontaneous mutation is errors of DNA replication and that the cell possesses multistep mechanisms to correct such errors (1). In addition, certain types of spontaneous DNA damage would cause mutations (2) and the cell comes equipped with mechanisms to repair such damage.Among 15 known E. coli mutator genes, 12 are shown to be involved in correction of replicational errors and/or spontaneous DNA damage (1, 3-6). Recently we obtained evidence that the mutT mutator gene is involved in a hitherto unknown mechanism for reducing spontaneous mutation frequency (7).A mutT mutator mutant shows a frequency of A-T -COG transversion 100-10,000 times the level of the wild type (8).The MutT protein specifically prevented misincorporation of dGMP onto poly(dA)/oligo(dT)20 template DNA in vitro (9).This antimutagenic effect of MutT protein appeared to be catalytic and was achieved through its action on dGTP but not on DNA or DNA polymerase (H.M. and M.S., unpublished results). We and others noted that the MutT protein has a weak nucleoside triphosphatase activity with a substrate preference to dGTP (9, 10). Subsequently we found that the nucleotide that is misincorporated opposite the dA residue of the template is not dGMP but rather its oxidized form, 8-oxodGMP. When 8-oxodGTP was added to an in vitro DNA replication system, 8-oxodGMP was incorporated opposite dA and dC residues of the template, with almost e...

MutT‐related error avoidance mechanism for DNA synthesis

“…The molecular weight of the protein, calculated from the predicted amino acid sequence, was 17 900, a value close to that estimated from analysis of SDS-PAGE (Mo et al 1992). When the amino acid sequences of the peptide fragments, derived from the purified 8-oxodGTPase protein, were aligned with the deduced amino acid sequence, there was a complete match.…”

Bioavailability and efficiency of rutin as an antioxidant: a human supplementation study

“…A dramatic decrease in oxidized pyrimidines (endogenous oxidative damage) occurred in both the placebo and rutin-treated subjects during the 6 weeks of the trial (Figure 5), suggesting the involvement of some other Rutin as an antioxidant SP Boyle et al dietary or seasonal factors in this protective effect. This effect was not apparent at the level of urinary 8OHdG (Table 2), which is believed by some to be a product of repair of oxidative damage to cellular DNA (Simic, 1992) or to the nucleotide DNA precursor pool (MO et al, 1992), or which may simply re¯ect oxidative stress (Lindahl, 1993). No correlation was found between the level of urinary 8OHdG exereted and the level of lymphocyte DNA damage detected either as endogenous strand breaks oxidized pyrimidine bases or resistance to H 2 O 2 challenge.…”

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

“…The E. coli MutT protein catalyzes the hydrolysis of 8-OH-dGTP [16], and A:TC:G transversions are induced with high frequency in mutT mutant strains [24,26,27], indicating the importance of 8-OH-dGTP as a source of mutations. Moreover, the presence of mammalian homologues of MutT (MTH1 proteins) supports this speculation [28]. Indeed, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice than wild-type mice [29].…”