The Dnmt3a gene, which encodes de novo-type DNA methyltransferase, encodes two isoforms, full-length Dnmt3a and Dnmt3a2, which lacks the N-terminal 219 amino acid residues. We found that Dnmt3a showed higher DNA-binding and DNA-methylation activities than Dnmt3a2. The N-terminal sequence from residues 1 to 211 was able to bind to DNA, but could not distinguish methylated and unmethylated CpG. Its binding to DNA was inhibited by a major groove binder. Four basic amino acid residues, Lys51, Lys53, Arg177 and Arg179, in the N-terminal region were crucial for the DNA-binding activity. The ectopically expressed N-terminal sequence (residues 1–211) was localized in nuclei, whereas that harbouring mutations at the four basic amino acid residues was also detected in the cytoplasm. The DNA-methylation activity of Dnmt3a with the mutations was suppressed under physiological salt conditions, which is similar that of Dnmt3a2. In addition, ectopically expressed Dnmt3a with mutations, as well as Dnmt3a2, could not be retained efficiently in nuclei on salt extraction. We conclude that the DNA-binding activity of the N-terminal domain contributes to the DNA-methyltransferase activity via anchoring of the whole molecule to DNA under physiological salt conditions.
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Research Article|
June 14 2011
Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a
Isao Suetake;
Isao Suetake
*Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Yuichi Mishima;
Yuichi Mishima
*Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Hironobu Kimura;
Hironobu Kimura
*Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Young-Ho Lee;
Young-Ho Lee
†Laboratory of Protein Folding, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Yuji Goto;
Yuji Goto
†Laboratory of Protein Folding, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Hideyuki Takeshima;
Hideyuki Takeshima
1
*Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Takahisa Ikegami;
Takahisa Ikegami
‡Laboratory of Structural Proteomics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Shoji Tajima
Shoji Tajima
2
*Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan
2To whom correspondence should be addressed (email tajima@protein.osaka-u.ac.jp).
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Publisher: Portland Press Ltd
Received:
February 07 2011
Revision Received:
April 11 2011
Accepted:
April 21 2011
Accepted Manuscript online:
April 21 2011
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2011 Biochemical Society
2011
Biochem J (2011) 437 (1): 141–148.
Article history
Received:
February 07 2011
Revision Received:
April 11 2011
Accepted:
April 21 2011
Accepted Manuscript online:
April 21 2011
Citation
Isao Suetake, Yuichi Mishima, Hironobu Kimura, Young-Ho Lee, Yuji Goto, Hideyuki Takeshima, Takahisa Ikegami, Shoji Tajima; Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a. Biochem J 1 July 2011; 437 (1): 141–148. doi: https://doi.org/10.1042/BJ20110241
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