Table 2:
Steady-state kinetics parameters for single nucleotide insertion of mismatch dAMP opposite unmodified dC or 11-mer peptide (NH2-GGGKGLGKGGA) conjugated to C5 position of cytosine (X) by hPol η. Catalytic efficiency values for unmodified dC and DpC sequences are shown in the fifth column (kcat/Km). Average effect is the percentage difference between the catalytic efficiency values of each DpC and its corresponding control. Michaelis Menten curves were calculated by nonlinear regression analysis using one-site hyperbolic fits in Prism 4.0 (Graphpad Software, La Jolla, CA, USA). Error was calculated by standard deviation of the mean in Prism 4.0.
| Template | Vmax (μM/min) | Km (μM) | kcat (min−1) | kcat/Km (min−1/μM) | Average Effect (%) |
|---|---|---|---|---|---|
|
| |||||
| CCC | 4.99 ± 1.47 | 251 ± 171 | 1.50 ± 0.44 | 0.006 ± 0.004 | ---- |
| CXC | 1.79 ± 0.18 | 113 ± 35 | 0.54 ± 0.05 | 0.005 ± 0.002 | −16.7% |
|
| |||||
| CCG | 2.46 ± 0.62 | 147 ± 103 | 1.65 ± 0.41 | 0.011 ± 0.008 | ---- |
| CXG | 2.20 ± 0.22 | 178 ± 47 | 1.47 ± 0.15 | 0.008 ± 0.002 | −27.3% |
|
| |||||
| CCT | 5.01 ± 0.20 | 18 ± 3.8 | 2.26 ± 0.09 | 0.129 ± 0.029 | ---- |
| CfCT | 2.78 ± 0.24 | 24 ± 9.9 | 2.23 ± 0.19 | 0.094 ± 0.004 | −27.1% |
| CXT | 5.29 ± 0.23 | 27 ± 5.6 | 2.40 ± 0.10 | 0.088 ± 0.020 | −31.8% |
|
| |||||
| ACA | 2.74 ± 0.23 | 107 ± 26 | 0.60 ± 0.07 | 0.006 ± 0.001 | ---- |
| AXA | 6.28 ± 0.73 | 130 ± 44 | 1.88 ± 0.22 | 0.015 ± 0.005 | +150% |
|
| |||||
| GCA | 5.47 ± 0.45 | 41 ± 14 | 1.20 ± 0.09 | 0.025 ± 0.010 | ---- |
| GXA | 1.92 ± 0.07 | 67 ± 8.6 | 0.42 ± 0.01 | 0.006 ± 0.001 | −76.0% |
|
| |||||
| TCA | 1.33 ± 0.06 | 30 ± 5.7 | 0.89 ± 0.02 | 0.030 ± 0.006 | ---- |
| TXA | 2.43 ± 0.27 | 246 ± 65 | 1.63 ± 0.08 | 0.007 ± 0.001 | −76.7% |
|
| |||||
| CCA | 4.63 ± 0.21 | 81 ± 12 | 2.08 ± 0.09 | 0.026 ± 0.004 | ---- |
| CXA | 4.86 ± 0.29 | 115 ± 21 | 2.19 ± 0.12 | 0.019 ± 0.003 | −26.9% |