Nucleotide sequence context influences HIV replication fidelity by modulating reverse transcriptase binding and product release
Division of Molecular Biology, Beckman Research Institute of the City of Hope Cancer Center, Duarte, CA, USA. Bioscience trends
(Impact Factor: 1.66).
An RNA template/DNA primer (T/P) complex derived from the env gene of HIV-1 was used to examine the kinetic effects of specific basepair substitutions on dNTP incorporation and RNase H cleavage by HIV-1 reverse transcriptase (RT). Single basepair substitutions 2 or 6 nucleotides upstream from a defined polymerization site (denoted -2 and -6) were engineered by oligonucleotide synthesis to provide 7 T/P substrates for kinetic analysis. A -6 A/T substitution in the wild type sequence resulted in 14- and 7-fold increases in the apparent second order rate constants (k(2app)) for U/A and U/G basepair formation. The k(2app) for U/A formation was relatively unchanged for all other T/P basepair changes. The -6A/T substitution also uniquely lowered the RNase H cleavage rate by 3-fold. Combined kinetic and thermodynamic analyses indicated that these effects were due almost exclusively to increases in the KD (k(off)/k(on)) of initial binding of RT to the T/P and the rate of product release. The data suggest that certain sequence contexts may influence RT fidelity by modulating enzyme binding/dissociation rather than by altering dNTP binding affinity or the rate of the bond forming step.
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