Effect of A and B metal ion site occupancy on conformational changes in an RB69 DNA polymerase ternary complex

Rapid chemical quench assays, as well as equilibrium and stopped-flow fluorescence experiments, were performed with an RB69 DNA polymerase (RB69 pol)-primer-template (P/T) complex containing 2-aminopurine (dAP) and a metal exchange-inert Rh(III) derivative of a deoxynucleoside triphosphate (Rh.dTTP). The objective was to determine the effect of catalytic metal ion (A site) occupancy on the affinity of an incoming Rh.dTTP for the RB69 pol-P/T binary complex and on the rate of the conformational change induced by Rh.dTTP binding. With Ca(2+) in the A site, the affinity of the incoming Rh.dTTP for the RB69 pol-P/T binary complex and the conformational change rate can be determined in the absence of chemistry. When Mg(2+) was added to a ternary complex containing Rh.dTTP opposite dAP, the templating base, nucleotidyl transfer occurred, but the rate of product formation was only one-tenth of that found with Mg.dTTP, as determined by rapid chemical quench assays. Rates of conformational change subsequent to formation of a ternary complex, in the absence of chemistry, were estimated from the rate of change in dAP fluorescence with an increase in the Rh.dTTP concentration. We have shown that there is an initial rapid quenching of dAP fluorescence followed by a second phase of dAP quenching, which has nearly the same rate as that of dTMP incorporation, as estimated from rapid chemical quench experiments. We have also demonstrated that the affinity of Rh.dTTP for occupancy of the B metal ion site is dependent on the presence of Ca(2+). However, a saturating Rh.dTTP concentration in the absence of Ca(2+) results in full quenching of dAP fluorescence, whereas a saturating Ca(2+) concentration in the absence of Rh.dTTP gives only partial quenching of dAP fluorescence. The implications of these results for the mechanism of Fingers closing, metal ion binding, and base selectivity are discussed.