title: Nucleotide modification at the gamma-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase.
abstract: The mechanism by which HIV-1 reverse transcriptase (HIV-RT) discriminates between the correct and incorrect nucleotide is not clearly understood. Chemically modified nucleotides containing 1-aminonaphthalene-5-sulfonate (ANS) attached to their gamma-phosphate were synthesized and used to probe nucleotide selection by this error prone polymerase. Primer extension reactions provide direct evidence that the polymerase is able to incorporate the gamma-modified nucleotides. Forward mutation assays reveal a 6-fold reduction in the mutational frequency with the modified nucleotides, and specific base substitutions are dramatically reduced or eliminated. Molecular modeling illustrates potential interactions between critical residues within the polymerase active site and the modified nucleotides. Our data demonstrate that the fidelity of reverse transcriptase is improved using modified nucleotides, and we suggest that specific modifications to the gamma-phosphate may be useful in designing new antiviral therapeutics or, more generally, as a tool for defining the structural role that the polymerase active site has on nucleotide selectivity.
authors.affiliation: Department of Biology and Biochemistry, University of Houston Houston, TX 77204-5001, USA.
authors.forename: Brent A
authors.initials: BA
authors.lastname: Mulder
authors.forename: Steve
authors.initials: S
authors.lastname: Anaya
authors.forename: Peilin
authors.initials: P
authors.lastname: Yu
authors.forename: Keun Woo
authors.initials: KW
authors.lastname: Lee
authors.forename: Anvy
authors.initials: A
authors.lastname: Nguyen
authors.forename: Jason
authors.initials: J
authors.lastname: Murphy
authors.forename: Richard
authors.initials: R
authors.lastname: Willson
authors.forename: James M
authors.initials: JM
authors.lastname: Briggs
authors.forename: Xiaolian
authors.initials: X
authors.lastname: Gao
authors.forename: Susan H
authors.initials: SH
authors.lastname: Hardin
mesh_terms: D001665:Binding Sites; D003854:Deoxyribonucleotides / Q000737:chemistry* / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009282:Naphthalenesulfonates / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry
chemical_list: D003854:Deoxyribonucleotides; D009282:Naphthalenesulfonates; D010710:Phosphates; D054303:HIV Reverse Transcriptase; C022042:1-naphthylamine-5-sulfonic acid