Table 1.
Summary of kinetic parameters for incorporation of non-natural nucleotides opposite non-instructional DNA lesions and thymine catalyzed by the exonuclease-deficient bacteriophage T4 DNA polymerasea
| DNA | Analog | Kd, app (µM) | kpol (s−1) | kpol/Kd, app (M−1 s−1) |
|---|---|---|---|---|
| Single abasic | dATPb | 35 ± 5 | 0.15 ± 0.01 | 4300 |
| Single abasic | 5-PhITPc | 14 ± 3 | 53 ± 4 | 3 800 000 |
| Single abasic | 5-F5-PhITP | 2.3 ± 0.7 | 15 ± 1 | 6 500 000 |
| Single abasic | 5-F4OMe-PhITP | 4 ± 1 | 18 ± 1 | 4 500 000 |
| Single abasic | 5-NITPd | 18 ± 3 | 126 ± 7 | 7 000 000 |
| Single abasic | 5-CITP | 172 ± 31 | 67 ± 5 | 390 000 |
| Single abasic | 5-MeCITP | 13.3 ± 0.9 | 79.8 ± 1.2 | 6 000 000 |
| Double abasic | dATPe | 157 ± 28 | 0.55 ± 0.05 | 3500 |
| Double abasic | 5-PhITPe | 12 ± 2 | 45 ± 3 | 3 750 000 |
| Double abasic | 5-F5-PhITP | 17 ± 4 | 10.6 ± 0.8 | 624 000 |
| Double abasic | 5-F4OMe-PhITP | 14 ± 3 | 30 ± 3 | 2 143 000 |
| Double abasic | 5-NITPe | 18 ± 5 | 70 ± 6 | 3 890 000 |
| Double abasic | 5-CITP | 172 ± 20 | 70 ± 4 | 407 000 |
| Double abasic | 5-MeCITP | 4.4 ± 0.5 | 82 ± 2 | 18 636 000 |
| Blunt end | dATPe | NDg | ND | ND |
| Blunt end | 5-PhITPe | 45 ± 8 | 0.007 ± 0.004 | 160 |
| Blunt end | 5-F5-PhITP | ND | ND | ND |
| Blunt end | 5-F4OMe-PhITP | ND | ND | ND |
| Blunt end | 5-NITPe | 21 ± 8 | 0.015 ± 0.004 | 710 |
| Blunt end | 5-CITP | ND | ND | ND |
| Blunt end | 5-MeCITP | 36.5 ± 9.9 | 0.047 ± 0.004 | 1290 |
| Thymine | dATPf | 10 ± 2 | 100 ± 10 | 10 000 000 |
| Thymine | 5-PhITPc | 25 ± 7 | 0.16 ± 0.01 | 6400 |
| Thymine | 5-F5-PhITP | 10 ± 2 | 0.072 ± 0.002 | 7000 |
| Thymine | 5-F4OMe-PhITP | 3.2 ± 0.6 | 0.062 ± 0.002 | 19 000 |
| Thymine | 5-NITPd | 9 ± 1 | 0.9 ± 0.1 | 100 000 |
| Thymine | 5-CITP | 818 ± 136 | 1.4 ± 0.1 | 1700 |
| Thymine | 5-MeCITP | 69 ± 26 | 7.0 ± 0.8 | 100 000 |
aAssays were performed using 500 nM gp43 exo−, 250 nM DNA substrates and variable concentrations of non-natural nucleotide in the presence of 10 mM Mg2+.
Kinetic values were taken from the following references: b(57), c(27), d(22), e(42) and f(35) for comparison.
gND: not determined as incorporation was too inefficient to measure accurately.