DNMT1 Enzymatic Assay Protocol

His-DNMT1 (182 kDa, human) was cloned, expressed and purified as described by Lee et al. [20] (link). The DNMT1 assay was performed according to Gros et al. [21] (link). Briefly, the reaction was started by addition of 90 nM of DNMT1 on a mix containing the tested compound (up to 1% DMSO), 1 µM of a AdoMet/[methyl-³H]-AdoMet mix in a ratio of 3-to-1 (isotopic dilution 1*:3) and 0.3 µM of biotinylated DNA duplex in 10 µL final volume. The reaction was incubated at 37°C for 2 h in reaction buffer (20 mM HEPES pH 7.2, 1 mM EDTA, 50 mM KCl, 25 µg/mL BSA). 8 µL are then transferred into a streptavidin-coated Flashplate PLUS (PerkinElmer) containing 190 µL of 20 µM AdoHcy (Sigma-Aldrich) in 50 mM Tris-HCl pH 7.4. The Flashplate was agitated at room temperature for 1 h, washed three times with 200 µL of 0.05% Tween-20 in 50 mM Tris-HCl pH 7.4 and read in 200 µL of 50 mM Tris-HCl pH 7.4 on TopCount NXT (PerkinElmer). Percentages of inhibition were calculated with the same formula as in DNMT3A assay.