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Abstract
The effect of DNA secondary structure on polymerization catalyzed by human immunodeficiency virus (HIV-1) reverse transcriptase (RT) was studied using a synthetic 66-nucleotide DNA template containing a stable hairpin structure. Four RT pause sites were identified within the first half of the hairpin stem. Additionally, five weak pause sites within the second half of the stem and the loop of the hairpin were identified at low temperatures. These weak pause sites were relocated to the site of the first few stem base pairs of two new hairpins formed due to a change in DNA secondary structure. Each pause site was correlated with a high free energy barrier of melting the stem base pair. Pre-steady state kinetic analysis of single nucleotide incorporation showed that polymerization at each pause site occurred by both a fast phase (10-20 s-1) and a slow phase (0. 02-0.07 s-1) during a single binding event. The reaction amplitudes of the fast phase were small (4-10% of enzyme sites), whereas the amplitudes of the slow phase were large (14-40%) at the pause sites. In contrast, only a single phase with a large reaction amplitude (32-50%) and a fast nucleotide incorporation rate (33-87 s-1) was observed at the non-pause sites. DNA substrates at all sites had similar dissociation rates (0.14-0.29 s-1) and overall binding affinity (16-86 nM). These results suggest that the DNA substrates at pause sites were bound in both productive and non-productive states at the polymerase site of RT. The non-productively bound DNA was slowly converted into a productive state upon melting of the next stem base pair without dissociation of the DNA from RT.
Highlights
Reverse transcriptase (RT)1 encoded by human immunodeficiency virus type 1 (HIV-1) is responsible for converting the viral single-stranded RNA genome into a double-stranded proviral DNA, which is integrated into the host genome [1]
A preincubated solution of wild-type HIV-1 RT and 5Ј-32P-labeled 25/66-mer in RT-Mg2ϩ buffer was mixed with all four deoxynucleotides in RT-Mg2ϩ buffer at either 37, 21, or 8 °C
HIV-1 RT could pause strongly at potential DNA secondary structures of the minus strand DNA template during plus strand synthesis in vivo since no accessory proteins possessing helicase-like activity have been found to increase the processivity of HIV-1 RT [15]
Summary
Proteins—Wild-type HIV-1 RT was prepared as described previously [15]. All concentrations of RT reported in this paper were determined spectrophotometrically at 280 nm, with an extinction coefficient of 260,450 MϪ1 cmϪ1. Wild-type RT (40 nM) was preincubated with increasing concentrations of DNA in RT binding buffer (50 mM Tris acetate, 10 mM magnesium acetate, 100 mM potassium acetate, 0.1 mM EDTA, pH 7.5 at 23 °C). Data from DNA dissociation rate measurements were fitted to a single exponential equation: [product] ϭ Ef ϩ ED(1 Ϫ exp(Ϫkt)), where Ef and ED represent the free and DNAbound active enzyme concentrations, respectively, in the preincubated. Data from the nitrocellulose-DEAE double-filter binding assay were fitted to a quadratic equation: [E1⁄7DNA] ϭ 0.5(Kd ϩ E0 ϩ D0) Ϫ 0.5((Kd ϩ E0 ϩ D0) Ϫ 4E0D0)1/2, where Kd represents the equilibrium dissociation constant for DNA substrate, E0 the active enzyme concentration, and D0 the DNA concentration. Mfold was based on the energy minimization method of Zuker [20], and the energy values were developed by SantaLucia et al [21]
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