Catalytic mechanism of DNA-(cytosine-C5)-methyltransferases revisited: Covalent intermediate formation is not essential for methyl group transfer by the murine Dnmt3a enzyme

Sabine Reither, Fuyang Li, Humaira Gowher, Albert Jeltsch

Research output: Contribution to journalArticlepeer-review

61 Scopus citations

Abstract

Co-transfections of reporter plasmids and plasmids encoding the catalytic domain of the murine Dnmt3a DNA methyltransferase lead to inhibition of reporter gene expression. As Dnmt3a mutants with C→A and E→A exchanges in the conserved PCQ and ENV motifs in the catalytic center of the enzyme also cause repression, we checked for their catalytic activity in vitro. Surprisingly, the activity of the cysteine variant and of the corresponding full-length Dnmt3a variant is only two to sixfold reduced with respect to wild-type Dnmt3a. In contrast, enzyme variants carrying E→A, E→D or E→Q exchanges of the ENV glutamate are catalytically almost inactive, demonstrating that this residue has a central function in catalysis. Since the glutamic acid residue contacts the flipped base, its main function could be to hold the target base at a position that supports methyl group transfer. Whereas wild-type Dnmt3a and the ENV variants form covalent complexes with 5-fluorocytidine modified DNA, the PCN variant does not. Therefore, covalent complex formation is not essential in the reaction mechanism of Dnmt3a. We propose that correct positioning of the flipped base and the cofactor and binding to the transition state of methyl group transfer are the most important roles of the Dnmt3a enzyme in the catalytic cycle of methyl group transfer.

Original languageEnglish (US)
Pages (from-to)675-684
Number of pages10
JournalJournal of Molecular Biology
Volume329
Issue number4
DOIs
StatePublished - Jun 13 2003
Externally publishedYes

Keywords

  • 5-methylcytosine
  • Covalent complex
  • DNA methylation
  • Enzyme mechanism
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Structural Biology

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