Table 1.
Steady-state kinetics data for insertion of nucleoside triphosphates opposite single xDNA bases by the Klenow fragment of DNA Pol I (exo−).a
| template base | nucleoside triphosphate | Vmax (% min−1)b | Km (μM) | efficiency (Vmax/Km) | relative efficiency |
|---|---|---|---|---|---|
| xA | dATP | 0.0041 (0.0012) | 23 (10) | 1.9 × 102 | 2.4 x10−2 |
| xA | dCTP | 0.0035 (0.0005) | 9.9 (2.3) | 3.9 × 102 | 4.9 × 10−2 |
| xA | dGTP | 0.0046 (0.0019) | 43 (20) | 1.1 × 102 | 1.4 × 10−2 |
| xA | dTTP | 0.15 (0.01) | 18 (3) | 8.0 × 103 | 1 |
|
| |||||
| xC | dATP | 0.40 (0.04) | 4.6 (1.5) | 8.7 × 104 | 6.7 × 10−1 |
| xC | dCTP | 0.065 (0.004) | 4.1 (1.5) | 2.0 × 104 | 1.5 × 10−1 |
| xC | dGTP | 3.5 (0.2) | 28 (3) | 1.3 × 105 | 1 |
| xC | dTTP | 0.19 (0.01) | 17 (5) | 1.2 × 104 | 9.2 × 10−2 |
|
| |||||
| xG | dATP | 0.0034 (0.0004) | 13 (10) | 3.8 × 102 | 5.1 × 10−3 |
| xG | dCTP | 0.26 (0.06) | 8 (10) | 7.5 × 104 | 1 |
| xG | dGTP | 0.0046 (0.0011) | 20 (9) | 2.7 × 102 | 3.6 × 10−3 |
| xG | dTTP | 0.0035 (0.0022) | 15 (17) | 1.3 × 102 | 1.7 × 10−3 |
|
| |||||
| xT | dATP | 9.0 (0.3) | 50 (6) | 1.8 × 105 | 1 |
| xT | dCTP | 0.19 (0.03) | 160 (30) | 1.2 × 103 | 6.7 × 10−3 |
| xT | dGTP | 0.064 (0.008) | 880 (10) | 7.7 × 101 | 4.3 × 10−4 |
| xT | dTTP | 0.59 (0.03) | 17 (2) | 3.6 × 104 | 2.0 × 10−1 |
|
| |||||
| T | dATP | 36 (4) | 3.4 (1.4) | 1.1 × 107 | -- |
a
Conditions: 200 nM 23mer/28mer primer-template duplex and varied polymerase concentrations in a buffer containing 50 mM Tris•HCI (pH 7.5), 10 mM MgCI2, 50 ug/mL BSA and 1 mM dithiothreitol, incubated at 37°C in a reaction volume of 10 μL. Standard deviations (n=3–5) are given in parentheses.
b
Normalized for the lowest enzyme concentration used.