Table 3. Binding of 8-oxoG.C- and THF.C-containing DNA by wild-type and mutant hOgg1 proteins.
| hOgg1 protein | Binding of 8-oxoG.C [Kd (nM)] | Binding of THF.C [Kd (nM)] |
|---|---|---|
| Wild-type | 8.2 ± 0.9 | 2.4 ± 0.3 |
| H270A | >266a | 4.6 ± 0.7 |
| H270R | 36.3 ± 4.9 | 4.2 ± 1.1 |
| H270L | >266a | 14.8 ± 3.3 |
| Q315A | 57.3 ± 10.9 | 5.5 ± 0.8 |
| F319A | >266a | 2.2 ± 0.4 |
| K249Q | 11.8 ± 4.3 | 4.8 ± 1.6 |
DNA probes were 32P-labeled 34mer oligodeoxyribonucleotide duplexes (Table 1). Kd values were determined by EMSA as described in Materials and Methods. DNA concentration was 0.25 nM and the hOgg1 concentration range was 1.25–250 nM. Values are averages of three independent experiments ± SD.
aThe amount of DNA bound at the highest concentration of hOgg1 was less than 50% of the total DNA. Therefore, the Kd values cannot be determined. It should be noted that the binding curves of hOgg1 (H270A, H270L and F319A) with the 8-oxoG.C-containing DNA were similar to those obtained with control DNA where the 8-oxoG.C pair was replaced with a G.C pair within the range of concentrations used.