Enhanced in vivo repair of O(4)-methylthymine by a mutant human DNA alkyltransferase

Carcinogenesis. 2000 Jul;21(7):1397-402.

Abstract

The repair of O(6)-methylguanine (m(6)G) by human O(6)-alkylguanine-DNA alkyltransferase (hAGT) is approximately 5000-fold greater than that for O(4)-methylthymine (m(4)T). To evaluate each adduct's contribution to mutagenesis, we previously created a mutant hAGT with increased specificity for m(4)T in vitro. The mutant and wild-type (WT) hAGT have now been expressed in bacterial strains that allow for the specific detection of A:T-->G:C and G:C-->A:T mutations induced by m(4)T and m(6)G, respectively. After exposure to the mutagenic methylating agent, N-methyl-N'-nitro-N-nitrosoguanidine, A:T-->G:C substitutions were reduced >4-fold in cells expressing the mutant hAGT compared with 1. 1-fold for WT hAGT. G:C-->A:T substitutions were decreased >2.5-fold in cells expressing the mutant hAGT, whereas WT hAGT totally prevented G:C-->A:T mutations. These results demonstrate that the altered substrate specificity of hAGT observed in vitro also occurs in vivo, and that it is responsible for the observed differences in mutations.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA Damage
  • DNA Repair / physiology*
  • Enzyme Inhibitors / pharmacology
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Guanine / analogs & derivatives*
  • Guanine / metabolism
  • Guanine / pharmacology
  • Humans
  • Lac Operon
  • Methylnitronitrosoguanidine / toxicity
  • O(6)-Methylguanine-DNA Methyltransferase / antagonists & inhibitors
  • O(6)-Methylguanine-DNA Methyltransferase / genetics*
  • O(6)-Methylguanine-DNA Methyltransferase / metabolism*
  • Phenotype
  • Point Mutation
  • Substrate Specificity
  • Thymine / analogs & derivatives
  • Thymine / metabolism

Substances

  • Enzyme Inhibitors
  • O(6)-benzylguanine
  • Methylnitronitrosoguanidine
  • O-4-methylthymine
  • Guanine
  • O-(6)-methylguanine
  • O(6)-Methylguanine-DNA Methyltransferase
  • Thymine