Document Type

Article

Publication Date

3-4-2013

Publication Title

ChemBioChem

Abstract

This report describes the use of several isosteric non-natural nucleotides as probes to evaluate the roles of nucleobase shape, size, solvation energies, and π-electron interactions as forces influencing key kinetic steps of the DNA polymerization cycle. Results are provided using representative high- and low-fidelity DNA polymerases. Results generated with the E. coli Klenow fragment reveal that this high-fidelity polymerase utilizes hydrophobic nucleotide analogues with higher catalytic efficiencies compared to hydrophilic analogues. These data support a major role for nucleobase desolvation during nucleotide selection and insertion. In contrast, the low-fidelity HIV-1 reverse transcriptase discriminates against hydrophobic analogues and only tolerates non-natural nucleotides that are capable of hydrogen-bonding or π-stacking interactions. Surprisingly, hydrophobic analogues that function as efficient substrates for the E. coli Klenow fragment behave as noncompetitive or uncompetitive inhibitors against HIV-1 reverse transcriptase. In these cases, the mode of inhibition depends upon the absence or presence of a templating nucleobase. Molecular modeling studies suggest that these analogues bind to the active site of reverse transcriptase as well as to a nearby hydrophobic binding pocket. Collectively, the studies using these non-natural nucleotides reveal important mechanistic differences between representative high- and low-fidelity DNA polymerases during nucleotide selection and incorporation.

Comments

This research was funded by the National Institutes of Health (CA118408 to A.J.B.).

DOI

10.1002/cbic.201200649

Version

Postprint

Volume

14

Issue

4

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