FIGURE 2.

LCA inhibits Dnmt1 activity. A, shown is a schematic of the Gla I-coupled Dnmt assay. Oligonucleotide 8006 contains a single nonmethylated CpG dinucleotide (shown in red). The fully methylated product oligonucleotide is a substrate for the restriction endonuclease Gla I. Cleavage of the product oligonucleotide releases the 5′ fluorophore from the 3′ quencher and generates fluorescence. AdoHcy, S-adenosyl homocysteine. B, shown is time dependence of assays containing 10 nm oligonucleotide 8006, 10 μm AdoMet, and 1 nm RFTS-lacking Dnmt1 (amino acids 621–1600) in the presence of DMSO (black), 10 μm 5-azaC (green), 10 μm SGI-1027 (blue), and 10 μm LCA (red). The addition of SGI-1027 and LCA fully inhibit Dnmt1 activity, whereas 5-azaC has no effect on fluorescence generation. RFU, relative fluorescence units.