Distinct enzymatic properties of recombinant mouse DNA methyltransferases Dnmt3a and Dnmt3b.
Recombinant mouse Dnmt3a and Dnmt3b were expressed in sf9 cells and purified to near homogeneity. The purified Dnmt3a and Dnmt3b gave specific activities of 1.8 +/- 0.3 and 1.3 +/- 0.1 mol/h/mol enzyme towards poly(dGdC)-poly(dGdC), respectively, which were the highest among those reported. Dnmt3a or Dnmt3b showed similar K(m) values towards poly(dIdC)-poly(dIdC) and poly(dGdC)-poly(dGdC). The K(m) values for S-adenosyl-L-methionine were not affected by the methyl-group acceptors, poly(dI-dC)-poly(dIdC) and poly(dG-dC)-poly(dGdC). The results indicate that the enzymes are de novo-type DNA methyltransferases. Dnmt3a and Dnmt3b activities were inhibited by Mn(2+) and Ni(2+) and showed broad pH optima around neutral pH. Both enzymes were susceptible to sodium ions, which inhibited their activity at around physiological ionic strength. However, Dnmt3a was fully active at physiological potassium concentration, but Dnmt3b was not. Using designed oligonucleotides for the analysis of cytosine methylation, we demonstrated that, in addition to CpG, Dnmt3a methylated CpA but not CpT and CpC, and that Dnmt3b methylated CpA and CpT but scarcely CpC. The relative activity of Dnmt3b towards nonCpG sequences was higher than that of Dnmt3a. These differences in enzymatic properties of Dnmt3a and Dnmt3b may contribute to the distinct functions of these enzymes in vivo.
Suetake
Isao
I
Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871.
Miyazaki
Junko
J
Murakami
Chikako
C
Takeshima
Hideyuki
H
Tajima
Shoji
S
D000818:Animals; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000235:genetics / Q000378:metabolism*; D000090205:DNA Methyltransferase 3A; D006863:Hydrogen-Ion Concentration; D051379:Mice; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D018411:Spodoptera; D000097584:DNA Methyltransferase 3B
D002413:Cations, Divalent; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A; D011994:Recombinant Proteins; D012436:S-Adenosylmethionine; D012965:Sodium Chloride; D000097584:DNA Methyltransferase 3B; C000715535:Dnmt3a protein, mouse