Table 2. Steady-State Kinetic Parameters for One-Base Extension of Control and M1dG-Containing Templates by Human DNA Polymerase ηa.
| template | mismatch | Km (μM) | kcat (s−1 × 10−3) | kcat/Km (μM−1 s−1 × 10−3) | f (misinsertion frequency)b |
|---|---|---|---|---|---|
| dG | dG·dC | 0.8 ± 0.3 | 75 ± 7 | 90 | 1 |
| dG·dA | 5 ± 2 | 7.6 ± 0.6 | 1.4 | 0.02 | |
| dG·dT | 1.1 ± 0.2 | 24 ± 1 | 22 | 0.2 | |
| dG·dG | 3 ± 1 | 13 ± 1 | 4 | 0.04 | |
| M1dG | M1dG·dC | 0.26 ± 0.09 | 29 ± 2 | 110 | 1.2 |
| M1dG·dA | 1.2 ± 0.5 | 5.8 ± 0.6 | 4.8 | 0.053 | |
| M1dG·dT | 10 ± 5 | 3.5 ± 0.2 | 0.35 | 0.0032 | |
| M1dG·dG | |||||
| (dGTP) | 3 ± 1 | 37 ± 1 | 12.3 | 0.11 | |
| (dTTP) | 0.77 ± 0.06 | 83 ± 2 | 108 | 1.2 |
a
Human DNA polymerase η (1.6 nM) was incubated with 50 nM primer-templates pairing dC, dA, dT, or dG with control (dG) or M1dG-containing templates and varied concentrations of dGTP (or dTTP for the M1dG·dG pair) for 5 min. The initial velocities of incorporation of dNTP (next base addition) were determined and graphed against the concentration of dGTP to determine kcat and Km values.
b
The frequencies of misincorporation were calculated relative to insertion of dNTP following dC·dG primer-templates as the ratio (kcat/Km)mismatch/(kcat/Km)dG·dC.