Motions of the Fingers Subdomain of Klentaq1 Are Fast and Not Rate Limiting: Implications for the Molecular Basis of Fidelity in DNA Polymerases

Abstract

Various kinetic studies on nucleotide incorporation by DNA polymerases have established that a rate-limiting step occurs that is crucial in the mechanism of discrimination between correct versus incorrect nucleotide. Crystallographic studies have indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. However, there is no direct evidence to support this hypothesis. In order to investigate whether or not the open-to-closed conformational transition affecting the fingers subdomain is rate limiting, we have developed a fluorescence resonance energy transfer (FRET) system, which monitors motions of the fingers subdomain. We establish that the closing of the fingers subdomain is significantly faster than the kinetically determined rate-limiting step. We propose that the rate-limiting step occurs after the closing of the fingers subdomain and is caused by local reorganization events in the active site.