New Experimental Strategies for Estimating Rates of Conformational Changes in DNA Polymerases

Abstract

To explore the mechanism used by RB69 DNA polymerase (RB69 pol), several new experimental strategies were applied to determine rates of conformational changes by stopped-flow fluorescence (sfF). 2-Aminopurine, a fluorescent analog of adenine, was used in the template strand of primer-template (P/T) as a probe to detect conformational changes in an RB69 pol · P/T · dNTP complex as reflected in the extent of base stacking. When a ddP/T was employed, conformational changes could be detected after dNTP binding but before nucleotidyl transfer since this step cannot occur with a ddP/T. “Exchange-inert” metal ion complexes such as Rh(III)dNTP, were also used to determine conformational changes in the absence of chemistry when Mg2+ was not present in the reaction. Another approach involved using a P/T labeled with a cyanine dye (Cy3) appended at the 5′-end of the template strand. A single molecule fluorescence technique was developed for studying the kinetics and mechanism of nucleotide addition and adapted for RB69 pol. The bimolecular rate constant for the association of RB69 pol to P/T was ~2.2 X 107 M−1s−1. The maximum rates for two steps of conformational changes observed by sfF were 264±21 and 19.5±0.4 per second respectively with the second rate corresponding to the rate of nucleotidyl transfer as determined by chemical quench. The conformational change before chemistry, induced by dNTP binding, reflects the movement of DNA template as well as the closing of the fingers domain, which we have shown not be the rate-limiting step in RB69pol catalyzed primer-extension reactions. This research was supported by GM63276-04.