Table 3.
Steady-state kinetic parameters for hpol κ19-256 catalysis on G4 DNA and non-G4 DNA substrates prepared in NaCl
| kcat (min−1) | KM,dNTP (μM) | kcat/KM,dNTP (min−1 μM−1) | Relative activitya | |
|---|---|---|---|---|
| 13/42-mer: dCMP insertion opposite template dG −10 nt from G22 | ||||
| non-G4 DNA | 2.7 ± 0.2 | 4.1 ± 1 | 0.66 | - |
| G4 DNA | 2.5 ± 0.2 | 3.8 ± 1 | 0.66 | 100% |
| 14/42-mer: dGMP insertion opposite template dC −9 nt from G22 | ||||
| non-G4 DNA | 2.0 ± 0.2 | 51 ± 10 | 0.039 | - |
| G4 DNA | 2.3 ± 0.2 | 140 ± 20 | 0.016 | 41% |
| 18/42-mer: dAMP insertion opposite template dT −5 nt from G22 | ||||
| non-G4 DNA | 10 ± 0.9 | 13 ± 4 | 0.77 | - |
| G4 DNA | 6.6 ± 0.3 | 19 ± 3 | 0.35 | 45% |
| 19/42-mer: dCMP insertion opposite template dG −4 nt from G22 | ||||
| non-G4 DNA | 7.3 ± 0.2 | 15 ± 1 | 0.49 | - |
| G4 DNA | 3.0 ± 0.08 | 21 ± 2 | 0.14 | 29% |
| 20/42-mer: dGMP insertion opposite template dC −3 nt from G22 | ||||
| non-G4 DNA | 4.1 ± 0.1 | 2.2 ± 0.3 | 1.86 | - |
| G4 DNA | 4.4 ± 0.2 | 1.9 ± 0.4 | 2.31 | 121% |
| 21/42-mer: dCMP insertion opposite template dG −2 nt from G22 | ||||
| non-G4 DNA | 5.0 ± 0.2 | 24 ± 3 | 0.21 | - |
| G4 DNA | 6.0 ± 0.3 | 20 ± 2 | 0.30 | 143% |
| 22/42-mer: dAMP insertion opposite template dT −1 nt from G22 | ||||
| non-G4 DNA | 4.7 ± 0.3 | 2.7 ± 0.7 | 1.74 | - |
| G4 DNA | 3.1 ± 0.1 | 4.5 ± 0.8 | 0.69 | 41% |
| 23/42-mer: dCMP insertion opposite G22 | ||||
| non-G4 DNA | 6.4 ± 0.2 | 5.0 ± 0.5 | 1.28 | - |
| G4 DNA | 1.1 ± 0.05 | 7.7 ± 1 | 0.14 | 11% |
a
Relative activity was calculated as [(kcat/Km,dNTP)G4 DNA/(kcat/Km,dNTP)non-G4 DNA] × 100. 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.