Table 6. Mispair extension fidelity of wild-type (WT) and mutant RTs, as obtained using template-primer D2-47/PG5-25.
| Enzyme | Base pair at the 3′ end* | kcat (min−1) | Km (μM) | kcat/Km (μM−1·min−1) | Mispair extension ratio (fext)† |
|---|---|---|---|---|---|
| WT RT | A:C | 1.30±0.14 | 41.3±5.6 | 3.2×10−2 | 2.8×10−3 |
| A:A | 0.15±0.04 | 266±107 | 5.7×10−4 | 5.1×10−5 | |
| A:G | 0.19±0.04 | 513±130 | 3.7×10−4 | 3.3×10−5 | |
| A114S | A:C | 1.64±0.53 | 280±134 | 5.9×10−3 | 2.6×10−3 (0.9) |
| A:A | 0.098±0.034 | 319±121 | 3.1×10−4 | 1.3×10−4 (2.6) | |
| A:G | 0.112±0.056 | 415±142 | 2.7×10−4 | 1.2×10−4 (3.5) | |
| A114G | A:C | 0.78±0.03 | 858±348 | 9.1×10−4 | 2.6×10−3 (0.9) |
| A:A | 0.032±0.007 | 824±228 | 3.9×10−5 | 1.1×10−4 (2.2) | |
| A:G | 0.022±0.006 | 230±53 | 9.6×10−5 | 2.8×10−4 (8.3) |
* The first base corresponds to the template and the second base to the primer.
† fext=[kcat (mismatched)/Km (mismatched)]/[kcat (matched)/Km (matched)], where kinetic parameters for the incorporation of dTTP on matched template-primers are given in Table 1. Numbers in parentheses represent the relative increase in the mispair extension ratio shown by the mutant RT relative to the wild-type enzyme. Our analysis assumes that RTs bind with roughly equal affinity to the matched and mismatched template-primer ends [22,35].