Nathalie Rouleau, Luis Rafael Silva, Ilya Ryabov, Philippe Roby & Alexandre Marcil
Covalent modification of DNA through methylation is catalyzed by specific DNA methyltransferases (DNMTs). DNMT1, 3a and 3b are the best characterized mammalian enzymes of this class, with DNMT1 thought to be responsible for maintenance of the methylated state, and DNMT3a and 3b responsible for de novo DNA methylation. Changes in DNA methylation patterns alter gene expression that may lead to human diseases. Until recently, DNA methylation was thought to be an irreversible process that was active only in mitotic cells; however recent findings suggest that the reverse process, through active demethylation, may occur in both mitotic and postmitotic cells. This opens the door to the possibility of using DNMT inhibitors to modulate aberrant gene expression in a variety of quiescent cell types. We describe here the development of a high-throughput, nonradioactive bead-based assay for DNA methylation that is suitable for screening applications to identify new DNMT inhibitors. For this purpose, biotinylated oligonucleotide is incubated with the enzyme in the presence of the cofactor SAM, following which 5-methyl-cytosine residues are quantified in a homogenous bridging assay format. The activity of different DNMT preparations was compared using this approach. Purified DNMT1 showed higher activity than 3a and 3b for the methylation of the oligonucleotide substrate, however we noted significant differences between DNMT1 preparations from different commercial vendors. A signal-to-background ratio (S/B) of approximately 40 was obtained using as little as 10 nM of DNMT1 with intra-assay variation of 7%. Signal generation was dependent on the presence of SAM, and decreased using known DNMT inhibitors, demonstrating the specificity of the reaction. Overall, the results presented will demonstrate that this novel homogenous and nonradioactive DNMT assay could represent a powerful alternative to established assay technologies for measuring methyltransferase activity.
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