Table 4.
Kinetics of Fpg and T4 DNA polymerase processing of 8-oxoG within putative mutational hot- and coldspots
| 5′-CC(8-oxoG)CC | 5′-AA(8-oxoG)AA | 5′-TG(8-oxoG)AC | 5′-TG(8-oxoG)AT | |
|---|---|---|---|---|
| Sequence context | ||||
| Fpg cleavage | ||||
| Km, nM | 1.3 ± 0.17 | 17.8 ± 2.15 | 43.8 ± 9.15 | 44.4 ± 7.55 |
| kcat, min−1 | 6.0 ± 2.16 | 9.6 ± 1.16 | 7.8 ± 1.23 | 6.0 ± 0.83 |
| kcat/Km, (nM⋅min)−1 | 4.62 | 0.54 | 0.18 | 0.14 |
| Synthesis by T4 DNA Pol (3′ → 5′ exonuclease-free) | ||||
| Insertion of C opposite 8-oxoG | ||||
| Km, μM | 0.683 ± 0.094 | 0.332 ± 0.032 | ND | 0.11 ± 0.008 |
| Vmax, nMDNA⋅min−1 | 0.35 ± 0.006 | 0.59 ± 0.017 | ND | 0.8 ± 0.014 |
| Vmax/Km, nMDNA⋅(μMdNTP⋅min)−1 | 0.51 | 1.78 | ND | 7.3 |
| Insertion of A opposite 8-oxoG | ||||
| Km, μM | 8.87 ± 0.45 | 1.38 ± 0.07 | ND | 0.34 ± 0.024 |
| Vmax, nMDNA⋅min−1 | 0.66 ± 0.01 | 0.86 ± 0.014 | ND | 0.78 ± 0.014 |
| Vmax/Km, nMDNA⋅(μMdNTP⋅min)−1 | 0.07 | 0.62 | ND | 2.3 |
| Extension of C:8-oxoG pair | ||||
| Km, μM | 8.24 ± 0.56 | 1.01 ± 0.217 | ND | 0.911 ± 0.23 |
| Vmax, nMDNA⋅min−1 | 0.74 ± 0.015 | 0.72 ± 0.049 | ND | 0.5 ± 0.048 |
| Vmax/Km, nMDNA⋅(μMdNTP⋅min)−1 | 0.09 | 0.71 | ND | 0.55 |
| Extension of A:8-oxoG pair | ||||
| Km, μM | 0.18 ± 0.006 | 0.102 ± 0.01 | ND | 0.32 ± 0.021 |
| Vmax, nMDNA⋅min−1 | 0.81 ± 0.002 | 0.72 ± 0.016 | ND | 0.66 ± 0.01 |
| Vmax/Km, nMDNA⋅(μMdNTP⋅min)−1 | 4.5 | 7.1 | ND | 2.1 |
| (Vmax/Km)inc·(Vmax/Km)ext | ||||
| C:8-oxoG pair | 0.05 | 1.26 | 4.02 | |
| A:8-oxoG pair | 0.32 | 4.4 | 4.83 |
ND, not determined.