FIGURE 4.

Glycosylase assay of MutY. A, a representative urea gel showing cleavage of A opposite oxoG by MutY (left set of lanes) and lack of cleavage of C opposite oxoG under the same conditions. (right set of lanes) To test the glycosylase activity of the cross-linked complex used in crystallography, we cross-linked the strand containing oxoG to the protein and then added to it the ³²P-labeled complimentary strand (containing either A or C to pair with oxoG). The reaction was quenched at various time points by adding NaOH into the mixture and heating at 90 °C for 15 min (see “Experimental Procedures” for detailed procedure). B, fraction of DNA cleaved in the two reactions in A plotted as a function of time. Each data point shows an average of three parallel experiments, and the error bar shows the standard deviation from the average. C, cleavage assays of catalytically active MutY (containing only the P164C mutation used in disulfide cross-linking) and the D144N mutant protein (also containing the P164C mutation) (neither of the two proteins is cross-linked to DNA in this experiment). During the course of the experiment, the active P164C MutY achieved almost 100% cleavage of the oxoG:A base pair, whereas no cleavage product was detected for the D144N mutant protein. Neither the catalytically active MutY nor the D144N mutant protein showed any detectable activity toward the anti-substrate oxoG:C.