FIGURE 2.
OGG1 complexed with 8-oxoG has guanine nucleotide exchange factor activity. A, fluorescent spectroscopic analysis of 8-oxoG binding to OGG1. FL indicates fluorescence. B, the Kd was calculated from the binding isotherms shown in A (18). C, interaction of OGG1 with Ras protein requires 8-oxoG base. H-Ras (2.7 pmol) was bound to nickel-agarose beads (see “Experimental Procedures”), and OGG1 ± 8-oxoG was added. After washing three times, the amount of OGG1 bound was analyzed by immunoblotting. D, OGG1 physically interacts with Ras protein. Increasing amounts (0.16–2.7 pmol) of His-H-Ras were immobilized on nickel-agarose beads before incubation with OGG1 (2.7 pmol) plus 8-oxoG (2.7 pmol) for 1 h at 4 °C. The proteins were stripped from the beads for immunoblotting. Upper panel, His-H-Ras bound to nickel-agarose beads; lower panel, OGG1 protein eluted from H-Ras. E, guanine nucleotides decrease the OGG1 interaction with Ras. His-H-Ras (2.7 pmol) bound to agarose beads was incubated with untagged OGG1 (2.7, 1.3, 0.67, and 0 pmol) and equimolar 8-oxoG for 30 min, and a 10-fold molar excess of GTP or GDP was then added for 30 min at 24 °C. Levels of eluted proteins were determined by Western blotting. F, exchange of Ras-bound GDP for GTP. H-Ras protein (6 pmol) was loaded with GDP (6 pmol), and nucleotide exchange was initiated by adding OGG1 (6 pmol) plus 8-oxoG (♦) or 0.6 pmol of OGG1 plus 8-oxoG (■), OGG1 (□), or 8-oxoG (▴) alone, together with GTPγS (60 pmol). G, GTP-GDP exchange by OGG1. H-Ras protein (6 pmol) was loaded with GTP, and guanine nucleotide exchange was initiated by adding 6 pmol of OGG1 plus 8-oxoG (♦) or 0.6 pmol of OGG1 plus 8-oxoG (■), OGG1 (□), or 8-oxoG (▴) alone, together with 10-fold excess GDP. Ras-GTP was quantified using pulldown immunoblot assays as in Fig. 1. G and F, right panels, bands in left panels were quantitated by densitometry (ImageJ), and the time course of nucleotide exchange is depicted graphically. n = 3–4.