FIGURE 5.

Determination of k_pol and K^dCTP_d with human REV1 for incorporation of dCTP opposite G and N²-BPG. A, a 10-fold excess of REV1 (300 nm) was incubated with 30 nm 24-mer/36-G-mer primer-template complex in the rapid quenched-flow instrument and mixed with increasing dCTP concentrations (▪, 20–1000 μm) to initiate the single turnover reactions. B, REV1 (240 nm) was incubated with 100 nm 24-mer/36-N²-BPG-mer primer-template complex in the rapid quenched-flow instrument and mixed with increasing dCTP concentrations (▪, 30–1000 μm) to initiate the reaction. Reactions were quenched with EDTA. A plot of observed rates of nucleotide incorporation (k_obs) versus [dCTP] was fit to a hyperbolic equation, as described under “Experimental Procedures.” A, G (unmodified): k_pol (maximal rate of nucleotide incorporation) = 0.87 ± 0.02 s^–1 and K ^dCTP_d = 40 ± 4 μm; B, N²-BPG: k_pol = 0.90 ± 0.02 s^–1 and K ^dCTP_d = 99 ± 9 μm.