title: 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.
authors: Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106 (USA).
authors: Edward A
authors: EA
authors: Motea
authors: Irene
authors: I
authors: Lee
authors: Anthony J
authors: AJ
authors: Berdis
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004256:DNA Polymerase I / Q000737:chemistry / Q000378:metabolism*; D004583:Electrons; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry / Q000201:enzymology*; D057927:Hydrophobic and Hydrophilic Interactions; D062105:Molecular Docking Simulation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D058105:Polymerization
chemical_list: D009711:Nucleotides; D004247:DNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D004256:DNA Polymerase I