title: Characterization of DNA-binding activity in the N-terminal domain of the DNA methyltransferase Dnmt3a.
abstract: 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.
authors.affiliation: Laboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
authors.forename: Isao
authors.initials: I
authors.lastname: Suetake
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Mishima
authors.forename: Hironobu
authors.initials: H
authors.lastname: Kimura
authors.forename: Young-Ho
authors.initials: YH
authors.lastname: Lee
authors.forename: Yuji
authors.initials: Y
authors.lastname: Goto
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeshima
authors.forename: Takahisa
authors.initials: T
authors.lastname: Ikegami
authors.forename: Shoji
authors.initials: S
authors.lastname: Tajima
mesh_terms: D001665:Binding Sites; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D006367:HeLa Cells; D006801:Humans; D017434:Protein Structure, Tertiary
chemical_list: C000717967:DNMT3A protein, human; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A