· P.J. Rothwell and G. Waksman.
A pre-equilibrium before nucleotide binding limits fingers subdomain closure by
Klentaq1
J. Biol. Chem, 282:28884-28892 (2007).
Numerous studies have been undertaken to
establish the mechanism of deoxyribonucleoside triphosphate (dNTP) binding and
template-directed incorporation by DNA polymerases. It has been established by
kinetic experiments that a rate-limiting step, crucial for dNTP selection,
occurs before chemical bond formation. Crystallographic studies indicated that
this step may be due to a large open-to-closed conformational transition
affecting the fingers subdomain. In previous studies we established a
Fluorescence Resonance Energy Transfer (FRET) system to monitor the open to
closed transition in the fingers subdomain of Klentaq1. By comparing the rates
of the fingers subdomain closure to that of the rate-limiting step for
Klentaq1, we showed that fingers subdomain motion was significantly faster than
the rate-limiting step. We have now used this system to characterise
DNA-binding as well as complete a more extensive characterisation of incorporation
of all four dNTPs. The data indicates that DNA binding occurs by a two step
association, and that dissociation of the DNA is significantly slower in the
case of the closed ternary complex (E’:p/t:dNTP). The data for nucleotide
incorporation indicates a step occurring before dNTP-binding which differs for
all four nucleotides. As the only difference between the (E:p/t) complexes is
the templating base, it would suggest an important role for the templating base
in initial ground state selection.