Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation

Steven S. Smith, Bruce E. Kaplan, Lawrence Sowers, Edward M. Newman

Research output: Contribution to journalArticle

110 Citations (Scopus)

Abstract

The properties of the methyl-directed DNA (cytosine-5-)-methyltransferase (EC 2.1.1.37) suggest that it is the enzyme that maintains patterns of methylation in the human genome. Proposals for the enzyme's mechanism of action suggest that 5-methyldeoxycytidine is produced from deoxycytidine via a dihydrocytosine intermediate. We have used an oligodeoxynucleotide containing 5-fluorodeoxycytidine as a suicide substrate to capture the enzyme and the dihydrocytosine intermediate. Gel retardation experiments demonstrate the formation of the expected covalent complex between duplex DNA containing 5-fluorodeoxycytidine and the human enzyme. Formation of the complex was dependent upon the presence of the methyl donor S-adenosylmethionine, suggesting that it comprises an enzyme-linked 5-substituted dihydrocytosine moiety in DNA. Dihydrocytosine derivatives are extremely labile toward hydrolytic deamination in aqueous solution. Because C-to-T transition mutations are especially prevalent at CG sites in human DNA, we have used high-performance liquid chromatography to search for thymidine that might be generated by hydrolysis during the methyl transfer reaction. Despite the potential for deamination inherent in the formation of the intermediate, the methyltransferase did not produce detectable amounts of thymidine. The data suggest that the ability of the human methyltransferase to preserve genetic information when copying a methylation pattern (i.e., its fidelity) is comparable to the ability of a mammalian DNA polymerase to preserve genetic information when copying a DNA sequence. Thus the high frequency of C-to-T transitions at CG sites in human DNA does not appear to be due to the normal enzymatic maintenance of methylation patterns.

Original languageEnglish (US)
Pages (from-to)4744-4748
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number10
StatePublished - 1992
Externally publishedYes

Fingerprint

Cytosine
Methyltransferases
Methylation
DNA
Enzymes
Deamination
5-methyldeoxycytidine
Aptitude
Thymidine
DNA (Cytosine-5-)-Methyltransferase
S-Adenosylmethionine
Deoxycytidine
Oligodeoxyribonucleotides
Human Genome
DNA-Directed DNA Polymerase
Suicide
Hydrolysis
Gels
High Pressure Liquid Chromatography
Maintenance

Keywords

  • DNA methylation
  • Hydrolytic deamination
  • Nucleophilic attack
  • Transition mutations

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Mechanism of human methyl-directed DNA methyltransferase and the fidelity of cytosine methylation. / Smith, Steven S.; Kaplan, Bruce E.; Sowers, Lawrence; Newman, Edward M.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 89, No. 10, 1992, p. 4744-4748.

Research output: Contribution to journalArticle

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