Table 8.
Kinetic parameters of extension reaction catalyzed by yeast Polζ from A:T or 3-dMeA:T base pair
Polζ (0.2 nm) was incubated with primer:template DNA substrate (10 nm) and increasing concentration of dNTPs for 10 min, at 37 °C. The nucleotide incorporation rate was plotted against dNTP concentration and the data were fit to the Michaelis-Menten equation. Apparent Km and kcat values were obtained from the fit and used to calculate the efficiency of deoxynucleotide incorporation (kcat/Km).
| Primer:template base pair | dNTP added | kcat | Km | kcat/Km | Relative catalytic efficiency |
|---|---|---|---|---|---|
| min−1 | μm | ||||
| A:T | dATP | 0.5 ± 0.012 | 0.04 ± 0.005 | 12.5 | 1 |
| 3-dMeA:T | dATP | 0.7 ± 0.07 | 1.7 ± 0.64 | 0.41 | 33 ↓ |