Table 1.
Kinetic parameters for the analogues, determined as described in Materials and Methods
| Template | Analog | Pol α | Klenow fragment | ||||||
|---|---|---|---|---|---|---|---|---|---|
| Vmax (% ext) | KM (μM) | V/K (% ext/μM) | Discrimination | Vmax (% ext) | KM (μM) | V/K (% ext/μM) | Discrimination | ||
| DNAA | d5OMeBTP | 1.7 (0.8) | 132 (79) | 0.013 | 396 | 25.8 (2.1) | 247 (38) | 0.104 | 703 |
| DNAG | d5OMeBTP | 1.0 (0.6) | 38 (29) | 0.026 | 322 | 89.0 (23.8) | 11 (3) | 8.0 | 17 |
| DNAC | d5OMeBTP | 6.4 (3.2) | 25 (4) | 0.26 | 88 | 14.4 (0.7) | 562 (179) | 0.0257 | 5770 |
| DNAT | d5OMeBTP | 2.0 (1.0) | 40 (17) | 0.050 | 187 | 39.2 (17) | 145 (65) | 0.271 | 480 |
| DNAA | d6OMeBTP | 0.85 (0.4) | 20 (12) | 0.044 | 119 | 21.9 (12.9) | 60 (38) | 0.37 | 200 |
| DNAG | d6OMeBTP | 0.85 (0.2) | 29 (10) | 0.029 | 279 | 10.0 (4.6) | 26 (14) | 0.39 | 351 |
| DNAC | d6OMeBTP | 8.2 (5.1) | 11 (6) | 0.72 | 32 | 13.2 (11.5) | 242 (242) | 0.0545 | 2720 |
| DNAT | d6OMeBTP | 3.3 (1.9) | 21 (15) | 0.15 | 62 | 25.4 (11.5) | 41 (12) | 0.62 | 211 |
| DNAA | d5F3BTP | 2.1 (1.1) | 27 (24) | 0.077 | 68 | 6.4 (1.1) | 79 (18) | 0.080 | 913 |
| DNAG | d5F3BTP | 1.9 (0.3) | 9 (4) | 0.21 | 39 | 61.5 (17.3) | 160 (68) | 0.386 | 355 |
| DNAC | d5F3BTP | 8.1 (2.5) | 9 (0.4) | 0.87 | 26 | 0.9 (0.3) | 67 (48) | 0.014 | 10500 |
| DNAT | d5F3BTP | 11.3 (3) | 17 (10) | 0.66 | 14 | 1.4 (0.2) | 37 (15) | 0.037 | 3530 |
| DNAA | d6F3BTP | 3.2 (2.5) | 31 (23) | 0.10 | 50 | 10.1 (1.3) | 75 (12) | 0.13 | 546 |
| DNAG | d6F3BTP | 2.7 (1.8) | 17 (15) | 0.15 | 53 | 89.5 (23.5) | 58 (33) | 1.5 | 89 |
| DNAC | d6F3BTP | 13 (4.2) | 19 (10) | 0.69 | 33 | 10.4 (2.7) | 263 (61) | 0.0394 | 3760 |
| DNAT | d6F3BTP | 15.9 (12.2) | 35 (28) | 0.45 | 21 | 24.3 (36.4) | 33 (13) | 0.73 | 179 |
| DNAA | dDNBTP | 3.4 (0.2) | 29 (1) | 0.12 | 45 | 1.4 (0.5) | 39 (15) | 0.036 | 2030 |
| DNAG | dDNBTP | 1.6 (0.3) | 3 (0.4) | 0.48 | 17 | 6.7 (3.6) | 81 (13) | 0.083 | 1650 |
| DNAC | dDNBTP | 15.6 (2.3) | 6 (5) | 2.5 | 9 | 1.6 (0.3) | 250 (15) | 0.0064 | 23100 |
| DNAT | dDNBTP | 5.7 (1.1) | 16 (12) | 0.36 | 26 | 1.6 (0.1) | 130 (5) | 0.012 | 10500 |
| DNAA | d5/6MeBTP | nd | nd | nd | nd | 16.8 | 169 | 0.099 | 740 |
| DNAG | d5/6MeBTP | nd | nd | nd | nd | 87.5 | 109 | 0.805 | 170 |
| DNAC | d5/6MeBTP | nd | nd | nd | nd | 20.5 | 1570 | 0.0131 | 11300 |
| DNAT | d5/6MeBTP | nd | nd | nd | nd | 13.8 | 111 | 0.124 | 1050 |
Discrimination is defined as the ratio of Vmax/KM of the cognate dNTP to that of the analogue. A Vmax of 1% extension corresponds to a kcat of 1 min−1 for pol α, and 0.83 min−1 for KF. Values are averages of at least three experiments; standard deviations are in parentheses. nd: not determined.