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

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish
Pages (from-to)3506-3512
Number of pages7
JournalNucleic acids research
Volume29
Issue number17
Publication statusPublished - Sep 1 2001
Externally publishedYes

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DNA (Cytosine-5-)-Methyltransferase
Methylation
Protein Isoforms
HEK293 Cells
Cytosine
Methyltransferases
DNA Methylation
Enzymes
poly(dC)
DNA

All Science Journal Classification (ASJC) codes

  • Genetics

Cite this

Aoki, A., Suetake, I., Miyagawa, J., Fujio, T., Chijiwa, T., Sasaki, H., & Tajima, S. (2001). Enzymatic properties of the de novo-type mouse DNA (cytosine-5) methyltransferases. Nucleic acids research, 29(17), 3506-3512.

Enzymatic properties of the de novo-type mouse DNA (cytosine-5) methyltransferases. / Aoki, Asako; Suetake, Isao; Miyagawa, Junichi; Fujio, Takayuki; Chijiwa, Takahito; Sasaki, Hiroyuki; Tajima, Shoji.

In: Nucleic acids research, Vol. 29, No. 17, 01.09.2001, p. 3506-3512.

Research output: Contribution to journalArticle

Aoki, A, Suetake, I, Miyagawa, J, Fujio, T, Chijiwa, T, Sasaki, H & Tajima, S 2001, 'Enzymatic properties of the de novo-type mouse DNA (cytosine-5) methyltransferases', Nucleic acids research, vol. 29, no. 17, pp. 3506-3512.
Aoki A, Suetake I, Miyagawa J, Fujio T, Chijiwa T, Sasaki H et al. Enzymatic properties of the de novo-type mouse DNA (cytosine-5) methyltransferases. Nucleic acids research. 2001 Sep 1;29(17):3506-3512.
Aoki, Asako ; Suetake, Isao ; Miyagawa, Junichi ; Fujio, Takayuki ; Chijiwa, Takahito ; Sasaki, Hiroyuki ; Tajima, Shoji. / Enzymatic properties of the de novo-type mouse DNA (cytosine-5) methyltransferases. In: Nucleic acids research. 2001 ; Vol. 29, No. 17. pp. 3506-3512.
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