Figure 7. The conformational-change step preceding phosphoryl transfer is a fidelity checkpoint.

(A) Complete kinetic mechanism for WT 3D^pol-catalyzed nucleotide incorporation in the presence of Mg²⁺. ER_n (3Dpol-sym/sub elongation complex); NTP (nucleotide); ER_nNTP (ternary complex); *ER_nNTP (activated ternary complex); *ER_n+1PP_i (activated product complex); ER_n+1PP_i (product complex); ER_n+1 (translocated 3D^pol-sym/sub product complex); PP_i (pyrophosphate). (B) AMP incorporation into sym/sub-U by WT 3D^pol (•) and G64S 3D^pol (○) in the presence of Mn²⁺. The solid lines represent the fit of the data to a single exponential with a k_obs of 27 ± 2 s⁻¹ for WT 3D^pol and 19 ± 1 s⁻¹ for G64S 3D^pol. 3D^pol (2 μM) was incubated with sym/sub (1 μM duplex) and rapidly mixed with ATP (1 mM) as described in Experimental Procedures. (C) Partial kinetic mechanism employed for simulation of the data shown below in panel D. (D) Accumulation of the activated ternary complex prior to phosphoryl transfer. Kinetics of AMP incorporation by WT 3D^pol and G64S 3D^pol in the presence of Mn²⁺ quenched by using either EDTA (•) or HCl (○). The solid line represents the simulation of the data to the mechanism shown in panel C with values for K_conf and k_chem shown in Table 2. EDTA-quench data reflects *ER_nNTP and *ER_n+1PP_i; HCl-quench data reflects *ER_n+1PP_i only. 3Dpol (2 μM) was incubated with sym/sub (1 μM duplex) and rapidly mixed with ATP (1 mM) as described in Experimental Procedures.