Detection of DNA Methylation by Dnmt3a Methyltransferase Using Methyl-Dependent Restriction Endonucleases
DNA methylation of cytosine residues in the CpG sites by DNA methyltransferases (MTases) is associated with various cellular processes. Eukaryotic MTase Dnmt3a is the key enzyme that establishes the de novo pattern of methylation. We have developed a new in vitro assay for DNA methylation by Dnmt3a based on the use of methyl-dependent restriction endonucleases (MD-RE) which specifically cleave methylated DNA. The catalytic domain of Dnmt3a (Dnmt3a-CD) was used together with KroI and PcsI MD-REs. The method consists of consecutive methylation and cleavage of fluorescently labeled DNA substrates. Reaction products were visualized in a polyacrylamide gel followed by the determination of the DNA methylation efficiency. Various substrates including partially methylated ones were tested for each endonuclease. The PcsI was found to be an optimal MD-RE. It recognizes two methylated CpG sites separated by seven nucleotide pairs, which roughly corresponds to the distance between the active centers of the Dnmt3a-CD tetramer. An optimal substrate was designed that contained two methylated cytosine residues and two target cytosines in the applicable for methylation by the Dnmt3a-CD orientation. The assay is reliable, simple, inexpensive and, unlike traditional methods, does not require the use of the radioactive compounds. The assay is suited to assess the effectiveness of Dnmt3a inhibitors as potential therapeutic agents and to determine the features of the Dnmt3a-CD functioning.