TABLE 2.
Ability of WT and AZT-resistant RTs to use various nucleotides as substrate acceptors for removal of ddAMP from blocked primer-templates through dinucleoside polyphosphate synthesisa
| HIV-1 RT |
kcat/Km (M−1 s−1)
|
|||||
|---|---|---|---|---|---|---|
| ATP | Me-ATP | UTP | Me-UTP | Dihydro-UTP | UDP | |
| WT | 0.21 ± 0.04 (1.0) | 0.22 ± 0.02 (1.0) | 0.19 ± 0.01 (1.0) | 0.17 ± 0.08 (1.0) | 0.056 ± 0.008 (1.0) | 0.32 ± 0.04 (1.0) |
| 215Y | 0.82 ± 0.06 (3.9)b | 1.3 ± 0.3 (5.9)b | 0.42 ± 0.10 (2.2)c | 0.59 ± 0.08 (3.5)b | 0.056 ± 0.003 (1.0)c | 0.23 ± 0.001 (0.7)c |
| 41L/215Y | 1.9 ± 0.4 (9.0)b | 3.0 ± 1.1 (14)b | 0.55 ± 0.03 (2.9)b | 0.98 ± 0.26 (5.8)b | 0.10 ± 0.002 (1.8)b | 0.29 ± 0.01 (0.9)c |
| 67N/70R/215Y/219Q | 3.3 ± 0.6 (16)b | 4.6 ± 2.3 (21)c | 1.4 ± 0.1 (7.4)b | 1.6 ± 0.6 (9.4)b | 0.19 ± 0.01 (3.4)b | 0.51 ± 0.01 (1.6)c |
Experiments were performed as described in the legend to Fig. 2B with the indicated RT and nucleotide substrate. The rate of dinucleoside polyphosphate synthesis was plotted against nucleotide concentration and fitted to a hyperbola using Sigmaplot 4.0 to obtain the kcat and Km. Data are the averages of two to eight experiments ± standard deviations. Numbers in parenthesis are increase (fold) versus the WT value. Significance of difference from WT was estimated by Student's t test corrected for multiple comparisons. N6-methyladenosine-5′-triphosphate (Me-ATP), 5-methyluridine-5′-triphosphate (Me-UTP), and dihydro-UTP were purchased from TriLink Biotechnologies, Inc. and treated with thermostable pyrophosphatase as described in the legend to Fig. 1, except dihydro-UTP, which was treated with inorganic pyrophosphatase (Roche Molecular Biochemicals) at 37°C to minimize breakdown to the diphosphate.
P < 0.05.
Not significantly different from WT value.