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Nucleic Acids Research, 2001, Vol. 29, No. 17 3506-3512
© 2001 Oxford University Press

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

Asako Aoki, Isao Suetake, Junichi Miyagawa, Takayuki Fujio, Takahito Chijiwa1, Hiroyuki Sasaki1 and Shoji Tajima*

Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan and 1National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan

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(dI-dC)-poly(dI-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, KmAdoMet, for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 µM when poly(dI-dC)-poly(dI-dC) was used, and 0.3, 1.2 and 0.8 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. The KmDNA values for Dnmt3a, Dnmt3b1 and Dnmt3b2 were 2.7, 1.3 and 1.5 µM when poly(dI-dC)-poly(dI-dC) was used, and 3.5, 1.0 and 0.9 µM when poly(dG-dC)-poly(dG-dC) was used, respectively. For the methylation specificity, Dnmt3a significantly methylated CpG >> CpA. On the other hand, Dnmt3b1 methylated CpG > CpT >= CpA. Immuno-purified Dnmt3a, Myc-tagged and overexpressed in HEK 293T cells, methylated CpG >> CpA > CpT. Neither Dnmt3a nor Dnmt3b1 methylated the first cytosine of CpC.

* To whom correspondence should be addressed. Tel: +81 6 6879 8627; Fax: +81 6 6879 8629; Email: tajima{at}protein.osaka-u.ac.jp


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