Figure 3.

Representative assays for glycosylase/lyase and glycosylase actvity of hOGG1. (A) Storage phosphor autoradiogram of the removal of FapyG opposite A by hOGG1 with a dye quench to determine the glycosylase/lyase activity. Reactions were performed at 37 °C, with a DNA concentration of 20 nM and a hOGG1 concentration of 100 nM (active site concentration). The first lane is a control without the enzyme, and the triangle indicates an increase in incubation time of up to 60 min. “S” and “P” refer to the bands arising from the substrate and product duplex, respectively. (B) Storage phosphor autoradiogram of the removal of FapyG opposite A by hOGG1 with a NaOH quench to determine the glycosylase activity. Reactions were performed at 37 °C, with a DNA concentration of 20 nM and a hOGG1 concentration of 100 nM (active site concentration). The first lane is a control without the enzyme, and the triangle indicates an increase in incubation time of up to 60 min. (C) Representative plot of the removal of FapyG from FapyG:A base-pair-containing duplex 1 by hOGG1 with a dye (red circles) or a NaOH quench (green inverted triangles). The graph was derived from the quantitation of the storage phosphor autoradiograms shown in panels A and B. The data were fit to a single exponential to determine k_gl or k_g. Rate constants from several experiments are averaged to provide the values in Table 1.