Figure 4

DNA glycosylase activities of EcMutY mutants. 1.8 fmol (0.18 nM) of 3'-end labeled A/G- (panel A) or A/GO- (panel B) containing 44-mer oligonucleotides were incubated with three concentrations (1, 10, and 50 nM based on Bradford assay) of EcMutY at 37°C for 30 min. With the active site titration assay upon binding with the A/GO-DNA, the active enzyme concentrations in the experiments are indicated as following. Lanes 1–3, wild-type (WT) EcMutY at 0.68, 6.8, and 34 nM were added. Lanes 4–6, V45A EcMutY at 0.48, 4.8, and 24 nM were added. Lanes 7–9, Q182L EcMutY at 0.25, 2.5, and 12.5 nM were added. Lanes 10–12, V45A/Q182L EcMutY at 0.4, 4, and 20 nM were added. After the reaction, the products were treated with 0.1 M NaOH and heated at 90°C for 30 min, followed by resuspension in formamide dye, and fractionated on a 14% denaturing sequencing gel. Arrows indicate the positions of intact oligonucleotide (I) and the nicked product (N).