Table 1. Error-prone Pol I achieves high levels of mutagenesis, with a preference for the targeted plasmid.
|
Mutagenesis*
|
Targeting†
|
|||
|---|---|---|---|---|
| Pol I | Initial | Optimized | Initial | Optimized |
| Wild type | 1.9 × 10-7 | 1.4 × 10-7 | 1 | 1 |
| D424A 1709N | 8.6 × 10-5 | 3.7 × 10-4 | 12.5 | 413 |
| A759R | 3.0 × 10-6 | ND | 14.2 | ND |
| D424A A759R | 5.9 × 10-5 | 7.9 × 10-5 | 4.9 | 68 |
| 1709N A759R | 3.6 × 10-4 | ND | 0.54 | ND |
| D424A1709N A759R | 7.0 × 10-4 | 2.1 × 10-3 | 1.0 | 390 |
ND, not determined.
*
By β-lactamase reversion assay.
†
The relative rate of plasmid versus chromosomal mutagenesis for each mutant polymerase was obtained after normalizing to that found for the wild-type polymerase. This factors in the different nature of the assays [forward assay (Rifr) versus reversion assay (β-lactamase)], and the difference in target numbers [single copy (Rifr) versus a multicopy (β-lactamase)].