ยท P.J. Rothwell, V. Mitaksov, and G.
Waksman.
Motions of the fingers subdomain of the Klentaq1 DNA polymerase I enzyme are
fast and not rate-limiting: implications for the molecular basis of fidelity in
DNA polymerases
Molecular Cell, 19:345-355 (2005).
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 re-organization events in the active site.