Fig. 1. Primer extension by wild-type and mutant RTs in assays containing a DNA template (D2-47) and lacking a complementary dNTP. Reactions were incubated for 5, 15, 30 and 60 min, as shown. Lanes marked with an asterisk indicate that all three nucleotides are included in the dNTP mix. The lanes marked with − A, − C and − T represent the missing nucleotide from the dNTP mix in the respective set of experiments. P and F indicate the positions of the unextended primer and the fully extended products, respectively. Prominent bands at positions + 4 and + 7 represent stop sites occurring after the incorporation of 4 and 7 nucleotides, respectively. Arrows are used to indicate bands representing specific stops produced by Val75 mutants. Boxes are used to highlight the differences between the wild-type RT and mutants V75I and V75T.

Fig. 2. Nucleotide concentration dependence on the first-order rates for correct (dTTP) or incorrect (dCTP and dGTP) incorporation into DNA/DNA 31/21-mer. (a) Pre-steady-state kinetics of nucleotide incorporation by wild-type HIV-1 RT. Preincubated solutions of RT (50 nM) and [5′-³²P]31/21-mer (100 nM) were mixed with increasing concentrations of nucleotide in a buffer containing 12 mM Mg²⁺ to start the reactions. The reactions were quenched at the indicated times and analyzed in polyacrylamide gels. The continuous lines represent the best fit of the data for dTTP incorporation to the burst equation (left panel). Center and right panels show the kinetics of dCTP and dGTP incorporation under single-turnover conditions (i.e., excess of RT over template–primer). In both cases, data were fit to a single-exponential equation. (b) The first-order rates measured above were plotted against the corresponding nucleotide concentration. The data were fit to a hyperbolic equation as described under Materials and Methods. The continuous line represents the best fit of the data to the hyperbolic equation. (c) Concentration dependence of dTTP (left), dCTP (centre) or dGTP (right) incorporation into the 31T/21P template–primer by mutant RTs V75A (●), V75F (▪) and V75I (). Incorporation rates (k_obs), obtained from the corresponding burst or single-exponential equations (not shown), were plotted against the nucleotide concentrations, and the data were fitted to the hyperbolic equation to obtain K_d and k_pol values. In all cases, kinetic parameters for correct nucleotide incorporations were determined in the presence of 12 mM Mg²⁺. Incorporation of dCTP and dGTP by mutants V75F and V75I was monitored in the presence of 18 mM Mg²⁺.

Fig. 3. Nucleotide concentration dependence on the first-order rates for dTTP incorporation into DNA/DNA 31/21-mers with G:T, G:G or G:A mismatches at their 3′ end. (a) Pre-steady-state kinetics of dTTP incorporation on a mismatched template–primer by wild-type HIV-1 RT. Preincubated solutions of RT (120 nM) and [5′-³²P]31/21-mer (100 nM) were mixed with increasing concentrations of nucleotide in a buffer containing 12 mM Mg²⁺ to start the reactions. The reactions were quenched at the indicated times and analyzed in polyacrylamide gels. The continuous lines represent the best fit of the data to the single-exponential equation. (b) The first-order rates (k_obs) measured above were plotted against the corresponding nucleotide concentration. The data were fitted to a hyperbolic equation as described under Materials and Methods. The solid lane represents the best fit of the data to the hyperbolic equation. (c) Concentration dependence of dTTP incorporation into the template–primers 31T/21PT (left), 31T/21PG (centre) and 31T/21PA (right) by mutant RTs V75A (●), V75F (▪) and V75I (). First-order rates, obtained from the corresponding equations (not shown), were plotted against the nucleotide concentrations, and the data were fitted to the hyperbolic equation to obtain K_d and k_pol values. Mispair extensions catalyzed by mutant V75A were carried out in the presence of a 12 mM Mg²⁺ concentration, while reactions performed with V75F or V75I were done in the presence of 18 mM Mg²⁺. Inhibition of the elongation reaction due to an excess of substrate was observed at dGTP concentrations above 12 mM with all RTs. This effect was not neutralized by increasing the concentration of Mg²⁺.

Fig. 4. Interactions between the peptide backbone of Val75 of HIV-1 RT and neighbouring residues. (a) View of the DNA polymerase site showing the location of the dsDNA template–primer (template strand in red and primer strand in white) and the incoming dNTP (yellow). Blue and green ribbon representations are used to indicate the location of the 66- and 51-kDa subunits of the RT, respectively. The β3–β4 hairpin loop (shown in orange) contains Val75, which is represented with a CPK model. Location of Val75 (yellow), Gln151 (blue) and template nucleotide + 1 (red) in the crystal structures of the ternary complex of HIV-1 RT·DNA/DNA·dTTP (PDB code 1RTD)³ (b), and binary complexes of HIV-1 RT and dsDNA (c and d). The structure shown in (c) (PDB code R0A) contains a 5′ template overhang of three nucleotides,⁴⁸ while in (d), the single-stranded template contains just one nucleotide (PDB code 2HMI).⁴⁶ Representative intramolecular distances (in angstroms) between peptide backbone groups in Val75 and neighbouring residues are indicated. Reported distances between the –CO– group of Val75 and the template nucleotide correspond to positions N3 (nitrogen base), C1′ (ribose), and the average distance to the five atoms in the ribose ring.

Fig. 5. Effect of RT mutations on the fidelity of DNA synthesis. Misincorporation efficiencies (a) and mispair extension efficiencies (b) for the wild-type HIV-1 RT and mutants V75A, V75F and V75I, as determined in steady-state kinetic assays (left) and in pre-steady-state kinetic assays (right).

Steady-state kinetic parameters for dTTP incorporation of wild-type (WT) and mutant RTs

D2-47/PG5-25 was used as the substrate. Data shown are the mean values ± SD obtained from a nonlinear least-squares fit of the kinetics data of a representative experiment to the Michaelis–Menten equation. Each of the experiments was performed independently at least twice. Interassay variability was below 20%.

Steady-state misinsertion fidelity of WT and mutant RTs, as obtained using the template primer D2-47/PG5-25

After formation of the RT-DNA/DNA complex, D2-47/PG5-25 elongation reactions were incubated at 37 °C for 20 s for dTTP, 45–90 s for dCTP and dGTP, and 60 min for dATP. Data shown are the mean values ± SD, obtained from a nonlinear least-squares fit of the kinetics data of a representative experiment to the Michaelis–Menten equation. Each of the assays was performed independently at least three times. Interassay variability of the kinetic parameters was below 20%.

Steady-state mispair extension fidelity of WT and mutant RTs, as obtained using template primer D2-47/PG5-25

After formation of the RT-DNA/DNA complex, mispair extension reactions were incubated at 37 °C for 20 s for the elongation of A:T and A:C mispairs, and 15–40 min for A:G and A:A mispairs. In all cases, we measured the incorporation of T opposite A at position + 1. Data shown are the mean values ± SD, obtained from a nonlinear least-squares fit of the kinetics data of a representative experiment to the Michaelis–Menten equation. Each of the assays was performed independently at least three times. Interassay variability of the kinetic parameters was below 20%.

Pre-steady-state kinetic parameters of WT and mutant RTs for misincorporation

The template–primer 31T/21P was used as the substrate. Data shown are the mean values ± SD of a representative experiment. Each of the assays was performed independently at least twice. Interassay variability was below 20%.

Pre-steady-state kinetic parameters of WT and mutant RTs for mispair extension

The template–primer 31T/21P was used as the substrate. Data shown are the mean values ± SD of a representative experiment. Each of the assays was performed independently at least twice. Interassay variability was below 20%.

Accuracy of WT and mutant RTs in M13mp2 lacZα forward and reversion mutation assays