Enzymatic properties of de novo-type mouse DNA (cytosine-5) methyltransferases

We have purified GST-fused recombinant mouse Dnmt3a and three isoforms, of mouse Dnmt3b to near homogeneity. Dnmt3b3, an isoform of Dnmt3b, did not ha ve DNA methylation activity. Dnmt3a, Dnmt3b1 or Dnmt3b2 showed similar acti vity toward poly(dG-dC)-poly(dG-dC) for measuring de novo methylation activ ity, and toward poly(dl-dC)-poly(dl-dC) for measuring total activity. This indicates that the enzymes are de novo-type DNA methyltransferases. The enz yme activity was inhibited by NaCl or KCl at concentrations > 100 mM. The k inetic 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 f or Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 muM when poly(dl-dC)-p oly(dl-dC) was used, and 3.5, 1.0 and 0.9 muM when poly(dG-dC)poly(dG-dC) w as used, respectively. For the methylation specificity, Dnmt3a significantl y methylated CpG much greater than CpA. On the other hand, Dnmt3b1 methylat ed CpG > CpT greater than or equal to CpA. Immuno-purified Dnmt3a, Myc-tagg ed and overexpressed in HEK 293T cells, methylated CpG much greater than Cp A > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.