Dynamics of nucleotide incorporation: snapshots revealed by 2-aminopurine fluorescence studies.


Formation of a noncanonical base pair between dFTP, a dTTP analogue that cannot form H bonds, and the fluorescent base analogue 2-aminopurine (2AP) was studied in order to discover how the bacteriophage T4 DNA polymerase selects nucleotides with high accuracy. Changes in 2AP fluorescence intensity provided a spectroscopic reporter of the nucleotide binding reactions, which were combined with rapid-quench, pre-steady-state reactions to measure product formation. These studies supported and extended previous findings that the T4 DNA polymerase binds nucleotides in multiple steps with increasing selectivity. With 2AP in the template position, initial dTTP binding was rapid but selective: K(d(dTTP)) (first step) = 31 microM; K(d(dCTP)) (first step) approximately 3 mM. In studies with dFTP, this step was revealed to have two components: formation of an initial preinsertion complex in which H bonds between bases in the newly forming base pair were not essential, which was followed by formation of a final preinsertion complex in which H bonds assisted. The second nucleotide binding step was characterized by increased discrimination against dTTP binding opposite template 2AP, K(d) (second step) = 367 microM, and additional conformational changes were detected in ternary enzyme-DNA-dTTP complexes, as expected for forming closed complexes. We demonstrate here that the second binding step occurs before formation of the phosphodiester bond. Thus, the high fidelity of nucleotide insertion by T4 DNA polymerase is accomplished by the sequential application of selectivity in first forming accurate preinsertion complexes, and then additional conformational changes are applied that further increase discrimination against incorrect nucleotides.



Kinetic Parameters, Nucleotide Analogs / Template Lesions, Fidelity, Biotech Applications, Methods, Nucleotide Incorporation

One line summary:

Highly accurate DNA polymerases, such as the T4 DNA polymerase, achieve high fidelity in nucleotide selection by using multiple binding steps with each successive step having increased stringency.


new topics/pols set partial results complete validated


Polymerase Reference Property Result Context
T4 D112AE114A Hariharan C2006 Kd 31uM Reaction: Nucleotide incorporation; Substrate: dTTP; Technique: Rapid quench (across template 2AP)

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