Table 7.
Steady-state kinetic parameters for hpol κ19-526 mis-insertion on G4 DNA and non-G4 DNA substrates prepared in LiCl
| kcat (min−1) | KM,dNTP (μM) | kcat/KM,dNTP (min−1 μM−1) | f ins a | |
|---|---|---|---|---|
| 18/42-mer: dCMP insertion opposite template dT (−5 nt from G22) | ||||
| non-G4 DNA | 0.13 ± 0.01 | 590 ± 100 | 0.00022 | 0.00016 |
| G4 DNA | 0.14 ± 0.01 | 580 ± 200 | 0.00024 | 0.00029 |
| 21/42-mer: dGMP insertion opposite template dG (−2 nt from G22) | ||||
| non-G4 DNA | 0.082 ± 0.005 | 190 ± 50 | 0.00043 | 0.0036 |
| G4 DNA | 0.10 ± 0.007 | 190 ± 50 | 0.00053 | 0.0048 |
| 23/42-mer: dGMP insertion opposite template dG (0 nt from G22) | ||||
| non-G4 DNA | 0.34 ± 0.02 | 570 ± 100 | 0.00060 | 0.0095 |
| G4 DNA | 0.31 ± 0.03 | 1300 ± 300 | 0.00024 | 0.0067 |
The mis-insertion frequency (fins) was calculated as (kcat/KM,incorrect dNTP)/(kcat/KM,correct dNTP). For determination of kinetic values, graphs of the initial rate of product formation versus dNTP concentration were plotted using non-linear regression analysis (one-site hyperbolic fit) in the GraphPad Prism program. The standard error of the fit is reported for each value.