Table 2.
Steady-state Kinetic Parameters for Human pol η-mediated Extension past Cyclo-dA and Cyclo-dG, and Unmodified dA and dG (designated with an X, and N is the nucleoside placed opposite X)a
| dNTP | kcat (min−1) | Km (μM) | kcat/Km (μM−1min−1) | f ext |
|---|---|---|---|---|
| X = (5'S)-cyclo-dA, N = dT | ||||
| dTTP | 0.93 ± 0.1 | 660 ± 80 | 0.0014 ± 0.0003 | 6.7 × 10−5 |
| dGTP | 3.3 ± 0.3 | 28 ± 2 | 0.12 ± 0.01 | 5.7 × 10−3 |
| dCTP | 6.3 ± 0.6 | 0.35 ± 0.03 | 21 ± 1 | 1 |
| dATP | 4.2 ± 0.4 | 15 ± 1 | 0.28 ± 0.01 | 1.3 × 10−2 |
| X = (5'S)-cyclo-dA, N = dA | ||||
| dTTP | 3.8 ± 0.3 | 42 ± 4 | 0.091 ± 0.009 | 8.3 × 10−1 |
| dGTP | 0.88 ± 0.1 | 710 ± 90 | 0.0012 ± 0.0002 | 1.1 × 10−2 |
| dCTP | 3.9 ± 0.3 | 35 ± 3 | 0.11 ± 1 | 1 |
| dATP | 0.62 ± 0.09 | 850 ± 100 | 0.00073 ± 0.00009 | 6.6 × 10−3 |
| X = dA, N = dT | ||||
| dTTP | 6.6 ± 0.6 | 19 ± 1 | 0.35 ± 0.03 | 5.9 × 10−3 |
| dGTP | 0.77 ± 0.09 | 830 ± 90 | 0.00093 ± 0.0001 | 1.6 × 10−5 |
| dCTP | 14 ± 1 | 0.24 ± 0.02 | 59 ± 5 | 1 |
| dATP | 6.4 ± 0.6 | 15 ± 1 | 0.43 ± 0.04 | 7.3 × 10−3 |
| X = (5'S)-cyclo-dG lesion-containing substrate, N = dC | ||||
| dTTP | 4.7 ± 0.4 | 34 ± 3 | 0.14 ± 0.01 | 8.8 × 10−3 |
| dGTP | 3.9 ± 0.4 | 38 ± 3 | 0.11 ± 0.01 | 6.8 × 10−3 |
| dCTP | 7.4 ± 0.7 | 0.46 ± 0.04 | 16 ± 1 | 1 |
| dATP | 5.4 ± 0.5 | 23 ± 2 | 0.24 ± 0.02 | 1.5 × 10−2 |
| X = (5'S)-cyclo-dG lesion-containing substrate, N = dT | ||||
| dTTP | 0.82 ± 0.1 | 840 ± 100 | 0.00098 ± 0.0002 | 9.8 × 10−1 |
| dGTP | 0.93 ± 0.2 | 690 ± 80 | 0.0013 ± 0.0003 | 1.3 |
| dCTP | 0.89 ± 0.1 | 880 ± 100 | 0.0010 ± 0.0002 | 1 |
| dATP | 3.3 ± 0.3 | 39 ± 3 | 0.085 ± 0.008 | 85 |
| X = dG, N = dC | ||||
| dTTP | 9.6 ± 0.9 | 36 ± 3 | 0.27 ± 0.02 | 4.8 × 10−3 |
| dGTP | 11 ± 1 | 24 ± 3 | 0.46 ± 0.04 | 8.2 × 10−3 |
| dCTP | 12 ± 1 | 0.21 ± 0.02 | 56 ± 5 | 1 |
| dATP | 4.1 ± 0.4 | 19 ± 1 | 0.22 ± 0.02 | 3.9 × 10−3 |
a
kcat and Km values were based on three independent measurements