Nucleotide modification at the ?-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase


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 ?-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 ?-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.




Copyright 2005 Nucleic Acids Research. Recommended citation: Mulder, Brent A., Steve Anaya, Peilin Yu, Keun Woo Lee, Anvy Nguyen, Jason Murphy, Richard Willson, James M. Briggs, Xiaolian Gao, and Susan H. Hardin. "Nucleotide modification at the ?-phosphate leads to the improved fidelity of HIV-1 reverse transcriptase." Nucleic acids research 33, no. 15 (2005): 4865-4873. doi: 10.1093/nar/gki779. URL: Reproduced in accordance with the original publisher's licensing terms and with permission from the authors.