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.
Motea, Edward A.; Lee, Irene; and Berdis, Anthony J., "Insights Into The Roles of Desolvation and π-Electron Interactions During DNA Polymerization" (2013). Chemistry Faculty Publications. 198.
This is the accepted version of the following article: Motea, E. A.; Lee, I.; Berdis, A. J. Insights into the Roles of Desolvation and π-Electron Interactions during DNA Polymerization. ChemBioChem 2013, 14, 489-498., which has been published in final form at http://onlinelibrary.wiley.com/wol1/doi/10.1002/cbic.201200649/abstract