FIG 5.
Steady-state kinetic analysis of the next base (dATP) insertion after 8-oxoG:C and 8-oxoG:A by wtDpo4 and mutant A181D. Dpo4 or mutant A181D (2 nM) and 20 nM DNA substrates (8-oxoG:C/8-oxoG:A) were used in the reaction mixture with various concentrations of dATP (0 to 200 μM). Incubation proceeded for 5 min at 37°C, and products formed were separated by PAGE. (A) dATP incorporation after 8-oxoG:C catalyzed by wtDpo4. (B) dATP incorporation after 8-oxoG:C by the mutant A181D. (C) dATP incorporation after 8-oxoG:A by wtDpo4. (D) dATP incorporation after 8-oxoG:A by the mutant A181D. kcat and Km values (estimated using the fit-to-hyperbolic-equations tool in Prism [GraphPad, San Diego, CA]) are listed in Table 4.