Figure 1. Single-turnover analysis of pyrophosphorolysis.

(a) Diagram illustrating the assay used to follow pyrophosphorolysis. A nicked DNA substrate utilizes PP_i to remove the 3′-[³²P]dCMP (C*) generating [α-³²P]dCTP (dCTP*). A cold dCTP trap was included in the reaction to prevent insertion of the radioactive product and to regenerate nicked DNA with an unlabeled 3′-terminus. Product formation (dCTP*) was monitored by TLC. (b) Data points, time, and ligand concentrations were selected to provide full coverage; i.e., multiple points were collected below and above reaction half-times (≥6 time points) and ligand binding affinities (≥5 concentrations), respectively. Time courses were fit to a single exponential (gray lines). (c) A secondary plot of the PP_i concentration dependence of the observed first-order rate constants (k_obs). These data were fit to a hyperbola (Eq. 1, gray line) to derive k_rev and K_d (Supplementary Table 1). (d) Simplified kinetic scheme for a DNA polymerase single-nucleotide insertion reaction. The chemical step (K₄) is flanked by enzyme conformational changes (K₃ and K₅). Ligand binding (K₁, K₂, K₆, and K₇) occurs to one form of the enzyme (circles) that undergoes a non-chemical conformational change to an alternate form (squares). These conformational states are often described as open or closed forms of the polymerase.