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Standard bandshift reactions (Fig. 3a) utilized 32 P-labelled`BH' probes that contained three CpG sites (underlined below) whose methylation/mismatch status was varied. The BH probes used in Fig. 3c were 59TCAGATTCGCGCZGGCTGCGATAAGCTGZGCGGATCCZGGGAATTCAGCT39 39AGTCTAAGCGCGGYCGACGCTATTCGACGYGCCTAGGGYCCTTAAGTCGA59 where Y C, m 5 C, T or U, and Z C or m 5 C (see underlined dinucleotides). The three CpG sites were identically modi®ed/mismatched within each probe. Binding reactions were carried out at room temperature for 30 min in 20 mM HEPES pH 7.9, 25 mM NaCl, 10 mM b-mercaptoethanol, 1 mM EDTA, 4% glycerol, 1% digitonin and 50 ng sonicated E. coli DNA. The glycosylase reaction does not occur under these conditions (data not shown). Complexes were electrophoresed through 6% polyacrylamide gels in 0:5 3 TBE at 4 8C.Complexes formed under conditions favourable to the glycosylase reaction (Fig. 3b, c) utilized the¯uorescent or 32 P-labelled JJ oligonucleotide (see above) as a probe. In standard gel-retardation reactions, 200 nM protein was incubated with 66 nM labelled oligonucleotide substrate and 333 nM unlabelled homoduplex oligonucleotide in 50 mM Tris±HCl pH 8.0, 1 mM DTT, 5% glycerol, 1 mM EDTA at 37 8C for 20 min. The samples were electrophoresed immediately through a 6% native 0:5 3 TBE polyacrylamide gel for 45 min at 100 V. A probe with an abasic site was generated by treatment of oligonucleotide JJ containing a MG×UG mismatch with the enzyme uracil DNA glycosylase.

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