Rhodium (III) derivative of dTTP and Ca2+ act interdependently to induce conformational changes in an RB69 DNA polymerase: primer/template complex

We report results from stopped‐flow fluorescence and chemical quench experiments involving RB69 DNA polymerase(E): primer‐template(D) complexes containing 2‐aminopurine (2AP) and the metal exchange‐inert Rhodium (Rh) (III) derivatives of deoxynucleoside triphosphates (N). With Ca 2+ present in the A site, no base incorporation occurs, so that the maximum conformational change rate (826s −1 ) can be determined independent of chemistry. Rates of conformational change subsequent to formation of a ternary complex but before chemistry were estimated from the rate of change in 2AP fluorescence with increasing [Rh(III)dTTP]. With Mg 2+ in the reaction, we found that the start point changed, thus the rate exceeded 1000s −1 . The maximum value of the subsequent rate (51.8 s −1 ) was consistent with the k pol value determined by chemical quench. Thus, the rate of the conformational change exceeds the rate of dTMP incorporation. We also demonstrate that occupancy and the affinity of Ca 2+ and Rh(III)dTTP for the A and B metal‐ion sites respectively, are interdependent. Using the scheme shown below, we found that the selectivity for correct versus incorrect dNTPs is determined by both their binding affinity (K d,overall = k −2 / (K 1 · k 2 )) and the reverse rate (k −2 ) by evaluation of the rate of the conformational change before chemistry. Scheme