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Kinetic Mechanism of Translocation and dNTP Binding in Individual DNA Polymerase Complexes
journal contribution
posted on 2013-06-19, 00:00 authored by Kate R. Lieberman, Joseph M. Dahl, Ai H. Mai, Ashley Cox, Mark Akeson, Hongyun WangComplexes
formed between phi29 DNA polymerase (DNAP) and DNA fluctuate
discretely between the pre-translocation and post-translocation states
on the millisecond time scale. The translocation fluctuations can
be observed in ionic current traces when individual complexes are
captured atop the α-hemolysin nanopore in an electric field.
The presence of complementary 2′-deoxynucleoside triphosphate
(dNTP) shifts the equilibrium across the translocation step toward
the post-translocation state. Here we have determined quantitatively
the kinetic relationship between the phi29 DNAP translocation step
and dNTP binding. We demonstrate that dNTP binds to phi29 DNAP–DNA
complexes only after the transition from the pre-translocation state
to the post-translocation state; dNTP binding rectifies the translocation
but it does not directly drive the translocation. Based on the measured
time traces of current amplitude, we developed a method for determining
the forward and reverse translocation rates and the dNTP association
and dissociation rates, individually at each dNTP concentration and
each voltage. The translocation rates, and their response to force,
match those determined for phi29 DNAP–DNA binary complexes
and are unaffected by dNTP. The dNTP association and dissociation
rates do not vary as a function of voltage, indicating that force
does not distort the polymerase active site and that dNTP binding
does not directly involve a displacement in the translocation direction.
This combined experimental and theoretical approach and the results
obtained provide a framework for separately evaluating the effects
of biological variables on the translocation transitions and their
effects on dNTP binding.