Table 6. Steady-State Kinetic Parameters for dNTP Incorporation Opposite 8-oxoG by Wild-Type and Variant hPols ι(1-445) in the Presence of 0.15 mM Mn2+.
pol ι(1–445) | dNTP | Km (μM) | kcat (s–1) | kcat/Km (s–1 mM–1) | finsa | relative efficiencyb |
---|---|---|---|---|---|---|
wild-type | A | 2.0 ± 0.3 | 0.015 ± 0.001 | 7.5 | 0.83 | |
T | 30 ± 2 | 0.046 ± 0.002 | 1.5 | 0.17 | ||
G | 0.90 ± 0.07 | 0.014 ± 0.0002 | 16 | 1.8 | ||
C | 4.2 ± 0.2 | 0.038 ± 0.001 | 9.0 | 1 | 1 | |
Δ1–25 | A | 17 ± 1 | 0.021 ± 0.0003 | 1.2 | 0.092 | |
T | 5.2 ± 0.3 | 0.028 ± 0.001 | 5.4 | 0.42 | ||
G | 0.21 ± 0.1 | 0.0082 ± 0.0004 | 39 | 3.0 | ||
C | 1.8 ± 0.2 | 0.023 ± 0.001 | 13 | 1 | 1.4 | |
ΔD17 | A | 2.0 ± 0.2 | 0.017 ± 0.0003 | 8.5 | 0.77 | |
T | 29 ± 1 | 0.055 ± 0.001 | 5.3 | 0.48 | ||
G | 0.53 ± 0.06 | 0.013 ± 0.0003 | 25 | 2.3 | ||
C | 3.5 ± 0.2 | 0.040 ± 0.0006 | 11 | 1 | 1.2 | |
R96G | A | 3.3 ± 0.3 | 0.0017 ± 0.00004 | 0.52 | 0.43 | |
T | 13 ± 1 | 0.0046 ± 0.0001 | 0.35 | 0.29 | ||
G | 1.8 ± 0.2 | 0.0038 ± 0.0001 | 2.1 | 1.8 | ||
C | 8.5 ± 0.5 | 0.010 ± 0.0002 | 1.2 | 1 | 0.13 | |
I261M | A | 1.4 ± 0.2 | 0.015 ± 0.0004 | 11 | 1.0 | |
T | 25 ± 3 | 0.026 ± 0.001 | 1.0 | 0.091 | ||
G | 0.66 ± 0.07 | 0.012 ± 0.0003 | 18 | 1.6 | ||
C | 3.1 ± 0.1 | 0.034 ± 1.0003 | 11 | 1 | 1.2 | |
E276K | A | 2.2 ± 0.3 | 0.022 ± 0.001 | 10 | 1.4 | |
T | 24 ± 4 | 0.044 ± 0.003 | 1.8 | 0.26 | ||
G | 0.60 ± 0.04 | 0.016 ± 0.0002 | 27 | 3.9 | ||
C | 3.2 ± 0.2 | 0.022 ± 0.0004 | 6.9 | 1 | 0.77 | |
Y374N | A | 1.4 ± 0.1 | 0.015 ± 0.0003 | 11 | 1.0 | |
T | 29 ± 6 | 0.025 ± 0.003 | 0.86 | 0.078 | ||
G | 0.72 ± 0.06 | 0.018 ± 0.0003 | 25 | 2.3 | ||
C | 3.6 ± 0.1 | 0.040 ± 0.0003 | 11 | 1 | 1.2 |
Misinsertion frequency, calculated by dividing kcat/Km for each dNTP incorporation by the kcat/Km for dCTP incorporation opposite 8-oxoG. All values are presented to two significant digits.
Relative efficiency, calculated by dividing kcat/Km of each pol ι(1–445) for dCTP incorporation opposite 8-oxoG by kcat/Km of wild-type pol ι(1–445) for dCTP incorporation opposite 8-oxoG.