Figure 5

In in vitro methyltransferase assay of hydralazine. There was no digestion with HpaII (lanes H, with 0 and 5 μM of hydralazine) indicating full methylation. Starting at 10 μM of hydralazine, bands in the lanes digested with HpaII (H) are visible, and at 20 μM the pattern of bands in HpaII (H) and MspI (M) digestions is similar indicating full demethylation. (MW: molecular weight). The substrate DNA for the in vitro methylation assay was a 1112 bp fragment of the type I Human Herpes Simplex virus tymidine kinase gene which has a high GC content. The methylation reaction contained 1 μg of substrate DNA and 10 units of M.SssI methylase (0.5 μmol/L, New England Biolabs, Beverly, MA) in a final volume of 30 μL. Hydralazine was added to final concentrations of 0, 5, 10, 15, and 20 μmol/L starting two hours before adding the M.SssI enzyme for the methylation reaction. Reactions were done at 37°C for 2 hours. After completion, the reaction the DNA was purified using the rapid PCR Purification system (Ijamsville, MD). Then, equal volumes of extracted DNA for each sample were placed in two separated eppendorf tubes for restriction enzyme digestion (one tube with HpaII and the other with MspI) both from New England Biolabs. Each reaction contained 10 U of the enzyme, in a final reaction volume of 50 μL at 37° for 3 hours. After digestion, samples were reduced to dryness by speedvac concentration and redissolved in 10 μL of ddH20 and then loaded into a 2% Tris-borate EDTA agarose gels for electrophoresis.