Table 1.
Kinetic Parameters for Nucleotide Incorporation Catalyzed by T4-exo− and L412M-exo− DNA polymerases
| Incorporation of dAMP opposite template T |
dTTP binding and incorporation of dTMP opposite template 2AP |
|||
|---|---|---|---|---|
| DNA polymerase | 2AP Fluorescence assaya | Rapid quench assayb | dTTP bindingc | dTMP incorporationd |
| T4-exo− | kunstack = 314 ± 18.5 s−1 | kproduct = 164.1 ± 5.4 s−1 | kbind = very rapid | kproduct = 165 ± 5 s−1 |
| Kd = 16 ± 3 μM | Kd = 17.2 ± 1.9 μM | Kd = 31 ± 4 μM | Kd = 367 ± 36 μM | |
| kchem = 345 s−1 | ||||
| L412M-exo− | kunstack = 177 ± 5 s−1 | kproduct = 117 ± 4.4 s−1 | Kd = 17 ± 3 μM | Kd = 252 ± 15 μM |
| Kd = 11 ± 1 μM | Kd = 8 ± 1.3 μM | |||
| kchem = 345 s−1 | ||||
a
The rates for base unstacking in the template strand and Kds, determined by the 2AP fluorescence assay, are from previous studies (15, 16).
b
The rates for product formation were determined under the same conditions as the 2AP fluorescence assays and are derived from the data presented in Figure 4.
c
The data for binding reactions with template 2AP and dTTP are from Figure 3. The rate of dTTP binding was determined in stopped-flow studies (15).
d
The kinetic parameters for dTMP incorporation opposite template 2AP are from 15.