Table 3.
Kinetic parameters for incorrect nucleotide incorporation (dGTP) into single-nucleotide gapped DNA (D-1) catalyzed by human Pol λ at 37 °C.
| Pol λ mutant |
kp (s−1) |
Kd (µM) |
kp/Kd (µM−1s−1) |
Kd ratioa |
Fidelityb |
|---|---|---|---|---|---|
| WTc | (4.0 ± 0.2) × 10−4 |
3.2 ± 0.5 | 1.3 × 10−4 | - | 8.3 × 10−5 |
| R386A | (1.55 ± 0.06) × 10−3 |
50 ± 10 | 3.1 × 10−5 | 16 | 3.6 × 10−4 |
| R386E | No observed incorporation | ||||
| R420A | (1.1 ± 0.2) × 10−4 |
800 ± 200 | 1.4 × 10−7 | 250 | 8.1 × 10−3 |
| K422A | (6.3 ± 0.3) × 10−4 |
8 ± 1 | 7.9 × 10−5 | 3 | 3.4 × 10−4 |
| Y505G | (1.23 ± 0.07) × 10−3 |
60 ± 10 | 2.1 × 10−5 | 19 | 2.0 × 10−5 |
| Y505A | (7.5 ± 0.2) × 10−4 |
30 ± 3 | 2.5 × 10−5 | 9 | 1.9 × 10−5 |
| F506A | (2.9 ± 0.3) × 10−4 |
19 ± 6 | 1.5 × 10−5 | 6 | 6.0 × 10−6 |
| A510E | (1.02 ± 0.03) × 10−2 |
15 ± 2 | 6.8 × 10−4 | 5 | 1.4 × 10−3 |
| R514A | (8.2 ± 0.8) × 10−4 |
80 ± 20 | 1.0 × 10−5 | 25 | 1.1 × 10−5 |
| R517A | (1.5 ± 0.1) × 10−3 |
1.0 ± 0.3 | 1.5 × 10−3 | 0.3 | 1.1 × 10−1 |
| R386A/A510E | (6.9 ± 0.6) × 10−3 |
170 ± 40 | 4.1 × 10−5 | 53 | 2.3 × 10−3 |
| R386A/R514A | (7 ± 1) × 10 4 |
320 ± 90 | 2.2 × 10−6 | 100 | 6.4 × 10−5 |
a
Calculated as (Kd) Mutant/( Kd )WT.
b
Calculated as (kp/Kd)Incorrect/[(kp/Kd)Correct + (kp/Kd)Incorrect].
c
The kinetic parameters for WT Pol λ are from the work of Fiala et al.12