In vitro DNA methylation assay

The methyl transfer activity assays were performed using the Promega bioluminescence assay (MTase-Glo) (Hsiao et al. 2016). The assay measures the methylation reaction by-product SAH, which is converted into ATP in a two-step reaction, and ATP was detected through a luciferase assay. The reaction in 30-μL volume contained 25 μM SAM, 5 μM substrate DNA (Fig. 1), and 0.5 μM individual protein or protein complex in 20 mM Tris-HCl (pH 7.5), 20% glycerol, 4 mM DTT, and 40 mM NaCl at 37°C. The individual protein or protein complex was preincubated with SAM for 5 min at room temperature before the addition of DNA. An aliquot of 10 μL of sample was taken from the reaction mixture and 0.1% (v/v) trifluoroacetic acid was added to stop the reaction. A 5-μL of reaction sample was transferred to a low-volume 384-well plate and the luminescence assay was performed according to the manufacturer's protocol. A Synergy 4 multimode microplate reader (BioTek) was used to measure luminescence signal. The kinetics measurements were performed for 1 h at 37°C with a total volume of 10 μL and varying concentration of substrate DNA.