Table 3. Kinetic parameters determined from fitting data in Figure 3 to the model for functional cooperativity as depicted in Figure 2.*.
| substrate | number of Dda molecules bound$ |
ku, s−1 | kd, s−1 |
|---|---|---|---|
| 12T16bp | 1 | 87 ± 6 | 5.6 ± 1.2 |
| 14T16bp | 2 | 65 ± 5 | 13 ± 2.0 |
| 21T16bp | 3 | 65 ± 5 | 9.5 ± 2.2 |
| 28T16bp | 4 | 76 ± 9 | 23 ± 7.3 |
| 12T20bp | 1 | 114 ± 5 | 17 ± 1.4 |
| 14T20bp | 2 | 110 ± 4 | 24 ± 1.5 |
| 21T20bp | 3 | 103 ± 6 | 23 ± 3.1 |
| 28T20bp | 4 | 116 ± 6 | 25 ± 3.4 |
Data were fit by using Kintek Global Kinetic Explorer (Kintek Corp.). Kinetic schemes were based on the model from Figure 2, which depicts two molecules bound to the substrate. Three or four unwinding steps were used for the 16bp and 20bp substrates, respectively. More complex kinetic mechanisms were used for substrates containing three or four Dda molecules (Supplementary data). Errors are standard errors obtained from the best fit of the data.
The number of Dda molecules bound per substrate is estimated from the binding site size and the length of the ssDNA overhang. In the case of the 12T substrates, the concentration of DNA substrate was in four-fold excess over the concentration of enzyme to ensure binding of one molecule of Dda per substrate molecule.