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

Asako Aoki, Isao Suetake, Junichi Miyagawa, Takayuki Fujio, Takahito Chijiwa, Hiroyuki Sasaki, Shoji Tajima

研究成果: ジャーナルへの寄稿学術誌査読

149 被引用数 (Scopus)

抄録

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 NaCI or KCI at concentrations >100 mM. The kinetic parameter, KmAdoMet, for Dnmt3a, Dnmt3b1 and Dnmt3b2 was 0.4, 1.2 and 0.9 μM when poly(dl-dC)-poly(dl-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(dl-dC)-poly(dl-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.

本文言語英語
ページ(範囲)3506-3512
ページ数7
ジャーナルNucleic acids research
29
17
DOI
出版ステータス出版済み - 9月 1 2001
外部発表はい

!!!All Science Journal Classification (ASJC) codes

  • 遺伝学

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