FIGURE 5.

Pre-steady-state kinetic analysis of hpol η^1–437 catalysis reveals that k_pol is four times greater in the presence of the WRN^{1–1092/E84A} HRDC-deletion mutant. A, pre-steady-state analysis of hpol η^1–437 (25 nm) catalyzed insertion of dCTP (1–250 μm; shown to the right of the plot) opposite dG in the presence of WRN^{1–1092/E84A} (500 nm). Product formation was plotted as a function of time and fit to Equation 2. B, the observed rate of product formation (k_obs) derived from panel A was plotted as a function of dCTP concentration for pre-steady-state experiments with hpol η^1–437 alone and in the presence of WRN^{1–1092/E84A}. Fitting the data to a hyperbolic equation yields an estimate of k_pol and K_d,dCTP: hpol η^1–437 alone (●): k_pol = 15.5 ± 3.0 s⁻¹ and K_d,dCTP = 28.5 ± 16.2 μm; hpol η^1–437 + WRN^{1–1092/E84A} (■): k_pol = 58.1 ± 5.2 s⁻¹ and K_d,dCTP = 31.8 ± 8.2 μm.