Fig. 2.

DME and ROS1 are 5-meC DNA glycosylases. (A) Double-stranded oligonucleotide substrates containing 5-meC (M) in CpG (CXGG) and non-CpG (XCGG, XAG, and AXT) sequence contexts or the corresponding unmethylated controls were incubated with purified DME (Upper) or ROS1 (Lower) as described in Materials and Methods. Reaction products were separated in a 12% denaturing polyacrylamide gel and visualized by autoradiography. (B) Release of 5-meC from methylated DNA. Plasmid DNA methylated with S-adenosyl-l-[methyl-³H]methionine (1,200 cpm) was incubated with purified DME or ROS1, and the released ethanol-soluble material was analyzed by two-dimensional TLC (see Materials and Methods). Distribution of radioactivity between thymine (filled bars) and 5-meC (open bars) was assayed by scintillation counting. The mean of duplicate experiments and their standard errors are shown.