Mechanism-based suppression of dideoxynucleotide resistance by K65R human immunodeficiency virus reverse transcriptase using an alpha-boranophosphate nucleoside analogue.

The amino acid change K65R in human immunodeficiency virus type 1-reverse transcriptase (RT) confers viral resistance to various 2',3'-dideoxynucleoside drugs in vivo. Using pre-steady state kinetic methods, we found that K65R-reverse transcriptase is 3.2-14-fold resistant to 2',3'-dideoxynucleotides in vitro relative to wild-type reverse transcriptase, in agreement with resistance levels observed in vivo. A decreased catalytic rate constant k(pol) mostly accounts for the lower incorporation efficiency observed for 2',3'-dideoxynucleotides. Examination of the crystal structure of the RT.DNA.dNTP complex suggested that both the charge at position 65 and the 3'-OH of the incoming nucleotide act in synergy during the creation of the phosphodiester bond, resulting in a more pronounced decreased catalytic rate constant for 2',3'-dideoxynucleotides than for dNTPs. This type of intramolecular activation of the leaving phosphate by the 3'-OH group appears to be conserved in several nucleotide phosphotransferases. These data were used to design dideoxynucleotide analogues targeting K65R RT specifically. alpha-Boranophosphate ddATP was found to be a 2-fold better substrate than dATP and inhibited DNA synthesis by K65R RT 153-fold better than ddATP. This complete suppression of drug resistance at the nucleotide level could serve for other reverse transcriptases for which drug resistance is achieved at the catalytic step.

Development of a new DNA sequencing method: 3'-ester cleavage catalyzed by Taq DNA polymerase.

A new 3'-esterified dTTP is incorporated into DNA by Taq DNA polymerase but does not act as a chain terminator. The esterase activity of the polymerase seems to be template dependent and occurs only if the next correct nucleotide is present.

Enhanced binding of azidothymidine-resistant human immunodeficiency virus 1 reverse transcriptase to the 3'-azido-3'-deoxythymidine 5'-monophosphate-terminated primer.

Human immunodeficiency virus type 1 is resistant to 3'-azido-3'-deoxythymidine (AZT) when four amino acid substitutions (D67N, K70R, T215F, and K219Q) are present simultaneously in its reverse transcriptase. Wild-type and AZT-resistant reverse transcriptases show identical binding to a 3'-azido-3'-deoxythymidine 5'-monophosphate (AZTMP)-terminated primer/RNA template. On DNA templates, the equilibrium dissociation constant (KD) for primer/template and AZT-resistant reverse transcriptase (RT) (KD = 4.1 nM) is similar to that of the wild-type enzyme (KD = 6.2 nM). However, koff is 4-25-fold lower for the AZT-resistant enzyme than for the wild-type enzyme, depending on the nucleotide and the template. The kinetic decay of a wild-type RT/primer/AZTMP-terminated DNA template complex is biphasic. Seventy percent of the initial complex decays with a rate constant greater than 0.05 s-1, and 30% with a rate constant of 0.0017 s-1. Decay of an AZT-resistant RT/AZTMP-terminated primer/DNA template complex is monophasic, with a rate constant of 0.0018 s-1. The last two nucleotides at the 3' end of the AZTMP-terminated DNA primer in complex with AZT-resistant RT, but not wild-type RT, and a DNA template are protected from exonuclease digestion, suggesting that enhanced binding of the 3' end of the AZTMP-terminated DNA primer to reverse transcriptase is involved in the mechanism of AZT resistance by human immunodeficiency virus type 1.