Table 4.
Steady-state kinetic analyses of nucleotide incorporation opposite undamaged A or 3-dMeA by human Polι
Polι (0.02–0.2 nm) was incubated with primer:template DNA substrate (10 nm) and increasing concentrations 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).
| Template nucleotide | Incoming nucleotide | kcat | Km | kcat/Km | Catalytic efficiency relative to T |
|---|---|---|---|---|---|
| min−1 | μm | ||||
| T | 10.7 ± 0.6 | 5.3 ± 0.9 | 2 | 1 | |
| A | A | 0.54 ± 0.02 | 220 ± 38.4 | 0.002 | 1 × 10−3 (↓ 1000 × ) |
| G | 0.48 ± 0.04 | 332 ± 70 | 0.0014 | 7 × 10−4 (↓ 1428 × ) | |
| C | ND | ND | |||
| T | 3.2 ± 0.09 | 12.4 ± 1.2 | 0.25 | 1 | |
| 3-dMeA | A | 1.5 ± 0.22 | 192 ± 96 | 0.0078 | 3.1 × 10−2 (↓ 32 × ) |
| G | 0.62 ± 0.02 | 59 ± 12.0 | 0.01 | 4 × 10−2 (↓ 25 × ) | |
| C | 0.37 ± 0.02 | 131.6 ± 29 | 0.0028 | 1.1 × 10−2 (↓ 89 × ) |