A Pre-equilibrium before Nucleotide Binding Limits Fingers Subdomain Closure by Klentaq1

Paul J Rothwell,Gabriel Waksman

doi:10.1074/jbc.m704824200

Paul J Rothwell, Gabriel Waksman

Open Access

https://doi.org/10.1074/jbc.m704824200

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Abstract

Numerous studies have been undertaken to establish the mechanism of 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 system to monitor the open-to-closed transition in the fingers subdomain of Klentaq1. By comparing the rates of the fingers subdomain closure with 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 characterize DNA binding as well as to complete a more extensive characterization of incorporation of all four dNTPs. The data indicate 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. The data for nucleotide incorporation indicate a step occurring before dNTP binding, which differs for all four nucleotides. As the only difference between the (E x p/t) complexes is the templating base, it would suggest an important role for the templating base in initial ground state selection.

Highlights

A basic model for DNA polymerases was proposed based on early kinetic work on T4 DNA polymerase [1], T7 DNA poly
After p/t binding, there follows an initial loose binding of the dNTP substrate. This initial ground state binding of the dNTP is used by some polymerases such as HIV-1 RT and T7 DNA polymerase, which are both replicative enzymes, to discriminate against non-complementary dNTPs by a factor of 250- and 390-fold, respectively [2, 4]
Binding of the correct nucleotide leads to a conformational change, which converts a loose E1⁄7p/t1⁄7dNTP ternary complex into a tight activated EЈ1⁄7p/t1⁄7dNTP complex capable of undergoing chemistry

Summary

Introduction

A basic model for DNA polymerases was proposed based on early kinetic work on T4 DNA polymerase [1], T7 DNA poly-. To monitor formation of the E1⁄7p/t complex, a solution of 20 nM pA488.6/t_G was mixed rapidly with a varying concentration of KTV649C(A594) (100 –1200 nM), and the signal produced from the decrease in the donor fluorescence of the labeled DNA was recorded.

Results

Conclusion