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A mutant HpaII methyltransferase functions as a mutator enzyme

J C Shen1, J M Zingg, A S Yang

  • 1Department of Biochemistry and Molecular Biology, USC/Norris Comprehensive Cancer Center, University of Southern California, Los Angeles 90033, USA.

Nucleic Acids Research
|November 11, 1995
PubMed
Summary

Researchers engineered DNA methyltransferase enzymes into mutator enzymes. These modified enzymes efficiently convert cytosine to uracil, causing mutations in living cells due to incomplete DNA repair.

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Area of Science:

  • Molecular Biology
  • Enzymology
  • Genetics

Background:

  • DNA methyltransferases (DNMTs) normally add methyl groups to DNA.
  • Under cofactor S-adenosylmethionine (AdoMet) limitation, DNMTs can act as C-->U mutator enzymes.
  • The HpaII methyltransferase's cofactor binding activity is crucial for its function.

Purpose of the Study:

  • To investigate if mutations inactivating cofactor binding in HpaII methyltransferase can create C-->U mutator enzymes.
  • To assess the mutagenic potential of these engineered enzymes in vivo.

Main Methods:

  • Site-directed mutagenesis was used to create HpaII methyltransferase variants (F38S and G40D) with impaired AdoMet binding.
  • In vitro assays measured cytosine deamination activity.
  • In vivo mutagenicity was assessed in bacterial systems with uracil-DNA glycosylase repair.

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Main Results:

  • Mutator enzymes F38S and G40D exhibited enhanced cytosine deamination in vitro and in vivo.
  • The generated G:U mispairs were not fully repaired by the bacterial uracil-DNA glycosylase repair pathway.
  • A potent mutator phenotype was observed in cells expressing these engineered enzymes.

Conclusions:

  • Mutations disrupting AdoMet binding can convert DNA methyltransferases into active C-->U mutator enzymes.
  • Engineered mutator enzymes demonstrate mutagenicity in living cells.
  • This study presents the first creation and characterization of mutator enzymes derived from DNA methyltransferases.