Figure 5

Time course studies of EcMutY mutant glycosylase activities. Panel A, A/G-containing DNA substrate (0.18 nM) was incubated with 49, 35, 18, and 29 nM of active site concentrations (all are 72 nM by Bradford assay) of WT, V45A, Q182L, and V45A/Q182L EcMutY, respectively, for various times at 37°C. Panel B is similar to panel A except that A/GO-containing DNA substrate (0.18 nM) was incubated with EcMutY for various times at 4°C. After reaction, the products were immediately frozen at -70°C in the presence of 0.1 N NaOH, heated at 90°C for 30 min, supplemented with 5 μl formamide dye, and loaded onto 14% 7 M urea sequencing gels. Numerical data were obtained from PhosphorImager quantitative analyses of gel images over three experiments. Percentages of DNA cleaved were plotted as a function of time.