Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: The enzyme modifies DNA in a non-processive manner and also methylates 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. coil 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 a ctivity of Dnmt3a is similar to that of Dnmt1: the K-m values for the unmet hylated and hemimethylated oligonucleotide substrates are 2.5 muM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively. The enzyme catal yzes the methylation of DNA in a distributive manner, suggesting that Dnmt3 a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we in vestigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sires, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificit y of Dnmt3a is completely different from that of the maintenance methyltran sferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated ov er unmethylated CpG sites and has almost no methylation activity at non-CpG sites. (C) 2001 Academic Press.