DNMT1 Assay and EMSA

A commercial DNMT Activity/Inhibition Assay (Active Motif, #55006) was used to quantify DNMT1 binding and activity. In this assay, we pre-incubated dsDNA or RNA/DNA hybrid with recombinant DNMT1, and then measured the methylation of CpG-rich sequences that were immobilized on 96-well plates. DNMT1 transfers the methyl group from S-Adenosyl methionine (SAM) supplied in the reaction to the immobilized DNA, and the methylated cytosine was quantified by ELISA with a specific methyl-binding protein and HRP-conjugated antibody. 500 ng of recombinant DNMT1 (Active Motif) was incubated with a titration of dsDNA or RNA/DNA hybrid at 25°C for 30 min in 10 μl binding buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl). Methylation level of CpG nucleotides by DNMT1 was measured on a spectrophotometer at 450 nm following manufacturer’s manual. Each reaction was carried out in triplicate. DNMT1 activity on dsDNA or RNA/DNA hybrid was calculated as Activity = 1-(OD₄₅₀ of sample with competitor - OD₄₅₀ background)/(OD₄₅₀ sample without competitor – OD₄₅₀ background). Average and SEM of triplicated assay were reported.

Electrophoretic mobility shift assay was carried out using biotinylated dsDNA or RNA/DNA hybrids with the sequence described above. Serial titrations of recombinant DNMT1 were incubated with 0.1 nM of biotinylated nucleotide substrates in DNMT1 binding buffer (5 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 1 mM DTT, 3% (v/v) glycerol, 100 mM NaCl) in a 10 μl reaction at 25°C for 30 min. For the competition assay, 100 nM of non-biotinylated dsDNA was added to the appropriate tubes and incubated for 15 min before biotinylated substrates were added. The 10 uL samples were mixed with 1.1 uL of Novex Hi-Density TBE Sample buffer (5X) and loaded to 6% DNA retardation gel (ThermoFisher, #EC6365BOX) and run at 100V in 0.5X TBE at 4°C for 1.5 hours. The gel was blotted to Hybond N⁺ nylon membrane in 0.5X TBE at 4°C at 15V for 17 hours. Biotin signal was detected using Chemiluminescent Nucleic Acid Detection Kit (ThermoFisher, #89880).