FIG. 5.

The methylation of substrate and/or competitor DNA has a minimal effect on MBD4 glycosylase activity. An approximately 50 nM concentration of a nonmethylated (A) or methylated (B) 164-bp 5S rRNA gene fragment was incubated with 25 nM MBD4 for 7.5 min in the presence of various amounts of competitor DNA. Reactions were run on a denaturing gel, and DNA species were detected and measured using a phosphorimager. Results are expressed as the percentage of cleavage, normalized to the degree of cleavage of the substrate without competitor DNA. Amounts of excess (n-fold) of competitor CpG sites relative to substrate CpG sites are indicated. Black bars, nonmethylated competitor; white bars, methylated competitor.