Abstract
BackgroundDnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.ResultsMutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.ConclusionsWe conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.
Highlights
Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals
We examined in detail the role of N167 (Fig. 1a) of Dnmt3a-CD that might be involved in positioning of the residues from motif VI [24]
DNA methylation activity DNA methylation activity of the purified enzymes was quantitatively assessed by calculating the initial rate of methylation (v0)
Summary
Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. DNA methylation is mediated and maintained by DNA methyltransferases (MTases). These enzymes transfer the methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) to the 5-position of cytosine mainly within CpG dinucleotides [5, 6]. Mammalian genomic DNA methylation is established de novo by DNA methyltransferases Dnmt3a and Dnmt3b [6,7,8]. The roles of the Dnmts are overlapping, and Dnmt contributes to de novo methylation as well as Dnmt enzymes are needed for the long term maintainance of DNA methylation [9]. Dnmt3-like protein (Dnmt3L) is a paralogue of the Dnmt enzymes that lacks catalytic activity and functions as regulatory protein of the Dnmt enzymes [10]
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