Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites.
We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase. Dnmt3a from mouse was cloned and expressed in Escherichia coli. It was shown to be catalytically active in E. coli cells in vivo. The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl. Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates. With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K(m) values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 microM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively. The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we investigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites.
Gowher
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Institut für Biochemie Fachbereich 8, Justus-Liebig-Universität, Heinrich-Buff-Ring 58, Giessen, 35392, Germany.
Jeltsch
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A
D000818:Animals; D002384:Catalysis; D018899:CpG Islands / Q000235:genetics; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D019175:DNA Methylation; D000090205:DNA Methyltransferase 3A; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009994:Osmolar Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
C000715535:Dnmt3a protein, mouse; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D004248:DNA (Cytosine-5-)-Methyltransferases; D000090205:DNA Methyltransferase 3A