Table 4.
Steady-state kinetic parameters for nucleotide incorporation by Exo- Klenow fragment and yeast polymerase η on 8-oxodG- and Tg-containing substratesa
| Substrates | dNTP | kcat (min-1) | km (nM) | kcat / Km (nM-1 min-1) | finc |
|---|---|---|---|---|---|
| 5′-(8-oxodG)-dT-3′ | By Exo-Klenow fragment with 15 mer Primer: 5′ - GCTAGGATCATAGCA - 3′ | ||||
| dATP | 0.082 ± 0.005 | (9.7 ± 0.2) × 102 | 8.5 × 10-5 | 5.7 × 10-2 | |
| dGTP | 0.082 ± 0.001 | (3.7 ± 0.2) × 104 | 2.2 × 10-7 | 1.5 × 10-4 | |
| dCTP | 0.038 ± 0.003 | 26 ± 7 | 1.5 × 10-3 | 1.0 | |
| dTTP | 0.016 ± 0.001 | (4.8 ± 0.7) × 105 | 3.3 × 10-8 | 2.2 × 10-5 | |
| By yeast polymerase η with 15 mer Primer: 5′ - GCTAGGATCATAGCA - 3′ | |||||
| dATP | 0.13 ± 0.01 | (6.9 ± 1.3) × 102 | 1.9 × 10-4 | 5.1 × 10-2 | |
| dGTP | 0.065 ± 0.010 | (4.0 ± 0.9) × 103 | 1.6 × 10-5 | 4.3 × 10-3 | |
| dCTP | 0.063 ± 0.005 | 17 ± 4 | 3.7 × 10-3 | 1.0 | |
| dTTP | 0.041 ± 0.005 | (1.3 ± 0.04) × 103 | 3.2 × 10-5 | 8.6 × 10-3 | |
| 5′-dG-Tg-3′ | By Exo- Klenow fragment with 14 mer Primer: 5′ - GCTAGGATCATAGC - 3′ | ||||
| dATP | 0.057 ± 0.005 | 36 ± 3 | 1.6 × 10-3 | 1.0 | |
| dGTP | 0.087 ± 0.014 | (2.1 ± 0.4) × 104 | 4.1 × 10-6 | 2.6 × 10-3 | |
| dCTP | 0.052 ± 0.005 | (1.1 ± 0.2) × 105 | 4.7 × 10-7 | 2.9 × 10-4 | |
| dTTP | 0.030 ± 0.003 | (2.0 ± 0.2) × 105 | 1.5 × 10-6 | 9.4 × 10-4 | |
| By yeast polymerase η with 14 mer Primer: 5′ - GCTAGGATCATAGC - 3′ | |||||
| dATP | 0.053 ± 0.001 | 71.1 ± 0.2 | 7.5 × 10-4 | 1.0 | |
| dGTP | 0.070 ± 0.005 | (6.0 ± 1.0) × 103 | 1.2 × 10-6 | 1.6 × 10-3 | |
| dCTP | 0.067 ± 0.002 | (4.0 ± 0.4) × 105 | 1.7 × 10-7 | 2.3 × 10-4 | |
| dTTP | 0.058 ± 0.001 | (1.1 ± 0.1) × 105 | 5.3 × 10-7 | 7.1 × 10-4 | |
a
kcat and Km are average values based on three independent measurements