We have purified GST-fused recombinant mouse Dnmt3a and three isoforms, of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not have DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar activity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activity, and toward poly(dl-dC)-poly(dl-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enzyme activity was inhibited by NaCl or KCl at concentrations > 100 mM. The kinetic parameter, K-m(AdoMet), for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 muM when poly(dl-dC)-poly(dl-dC) was used, and 0.3, 1.2 and 0.8 LM when poly(dG-dC)-poly(dG-dC) was used, respectively. The K-m(DNA) values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 muM when poly(dl-dC)-poly(dl-dC) was used, and 3.5, 1.0 and 0.9 muM when poly(dG-dC)poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG much greater than CpA. On the other hand, Dnmt3b1 methylated CpG > CpT greater than or equal to CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG much greater than CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.
