Insights into the Roles of Desolvation and π‐Electron Interactions during DNA Polymerization

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.

The use of modified and non-natural nucleotides provide unique insights into pro-mutagenic replication catalyzed by polymerase eta

“…We argue that rotational freedom of the nitro group improves its efficiency for incorporation. However, other features such as increased pi-electron density associated with the nitro moiety may also play a role as this has been previously demonstrated for high-fidelity DNA polymerases when replicating non-instructional DNA lesions ( 41 , 42 ).…”

RB69 DNA Polymerase Structure, Kinetics, and Fidelity

“…86 Several groups have made important contributions that have provided insights into this issue. 87−94 Kool’s group challenged Watson and Crick’s argument about the basis for nucleotide discrimination by using an array of nonpolar nucleoside analogues, such as 2′-deoxyribo-4-methylbenzimidazole (dZ), 2′-deoxyribo-2,4-difluorotoluene (dF), and 2′-deoxyribo-9-methyl-1 H -imidazole-[(4,5)-β]-pyridine (dQ), in primer extension assays to show that base pairing was not as important as shape for base selectivity. 86,95−97 The analogue that created the most controversy was dF, which was reported to be a nonpolar, non-hydrogen bonding isostere of dT.…”

A non-natural nucleotide uses a specific pocket to selectively inhibit telomerase activity

“…Modification of the nucleobase at these positions will additionally disturb proper Watson-Crick base pairing with the RNA template. Finally, the individual analogs investigated are already known to exhibit differential effects on diverse DNA polymerases that include HIV-1 RT, bacteriophage T4 DNA polymerase, and Escherichia coli Klenow fragment [32–36].…”