Net155h001mc

All methylation reactions were performed in a buffer containing 50 mM Tris–HCl pH 8, 50 mM NaCl and 200 μM MgSO₄. The following substrates were annealed in the same buffer at 2.5 μM concentration by heating primers to 100°C for 30 s and then cooling to room temperature on the bench top: dsDNA target (oTNM38, oTMN39); dsDNA non-target (oTMN40, oTMN41); and dsRNA (oJR270, oJR271). The H³-SAM (Perkin Elmer: NET155H001MC) was used at a concentration of 1 μM in solution. The purified DnmA, YabB, or DnmA (Y465A) was added to a concentration of 1 μM and all substrates were used at 0.25 μM in solution. The proteins were added in excess to determine if there was any off target methylation activity at higher protein concentrations. The total reaction solution came to 10 μl. All reactions were incubated at 37°C for 150 min unless otherwise specified. Reactions were stopped using 450 μl of 10% TCA and placed on ice. The samples were filtrated using Glass microfiber filters (GE: 1822-025), washed with cold 70% ethanol, dried, and placed in a scintillation counter to measure mmol incorporation.

The compounds OTS193320 or OTS186935 were serially diluted (10 doses) and mixed with biotin conjugated histone H3 peptide (1-21) (final conc. 350 nM) (Millipore: 12-403) and adenosyl-L-Methionine, S-[Methyl-³H]- (final conc. 100 nM) (Perkin Elmer: NET155H001MC) in the assay buffer (20 mM Tris pH 8.0, 10 mM MgCl₂, 50 mM NaCl, 10 mM DTT, 0.03% Tween-80). N-terminal GST fused SUV39H2 (final conc. 20 nM) (in-house) was added to start reaction and incubated at room temperature for 3 hours. The reaction was stopped by adding 2.5 mg/mL of Streptavidin SPA beads in bead buffer (20 mM Tris pH 8.0, 500 mM MgCl₂, 50 mM NaCl, 10 mM DTT, 0.03 % Tween-80). The radioactive signal was measured with Trilux-Microbeta counter (Perkin Elmer). IC₅₀ was calculated with XLfit.