TABLE 1.
DNA binding properties of RecQ in different nucleotide states
Shown are 25 °C, dT54, 0.5 μm RecQ Trp fluorescence stopped-flow data (except for Trp fluorescence emission spectra). Nomenclature of parameters refers to Fig. 1G. All parameters with dimensions involving concentration are specified based on DNA concentrations expressed in constituent nucleotide units (nt).
| Parameter | Source data | Nucleotide-free | AMPPNP | ADP | ADP-AlF4 |
|---|---|---|---|---|---|
| Total DNA-induced quench (%)a | Trp spectrab | 65 ± 4 | 69 ± 4 (5 ± 1) | 62 ± 4 (11 ± 1) | 70 ± 3 (17 ± 2) |
| Amplitudes | 59 ± 1 | 61 ± 1 | 51 ± 2 | 62 ± 2 | |
| Contribution of slow phase (% of total quench) | Amplitudes | 49 ± 7 | 56 ± 3 | 14 ± 10 | 53 ± 2 |
| DNA-binding stoichiometry (nt/RecQ) | Amplitudes | 20 ± 3 | 14 ± 8 | 14 ± 3 | 40 ± 18 |
| k1 (μm−1 s−1) | Binding kinetics | 7.8 ± 1.7 | 5.0 ± 0.6 | 9.1 ± 0.6 | 0.76 ± 0.07 |
| k–1 (s−1) | Binding kinetics | 140 ± 110 | 88 ± 19 | 140 ± 20 | 10 ± 7 |
| K1 (μm) | k−1/k1 | 18 ± 18 | 18 ± 6 | 15 ± 3 | 13 ± 10 |
| k2 + k−2 (s−1) | Binding kinetics | 8.5 ± 3.0 | 3.2 ± 0.4 | 18 ± 5 | 2.0 ± 0.3 |
| k−2 (s−1) | Binding kinetics | 2.2 ± 3.3 | 0.30 ± 0.50 | 6.7 ± 2.7 | < 0.1 |
| DxSO4 chasingc | 4.1 ± 0.3 | 0.40 ± 0.03 | 12 ± 1 | < 0.01 | |
| Heparin chasingd | 4.2 ± 0.8 | 0.89 ± 0.03e | 10 ± 2 | < 0.01 | |
| K2 | k2/k−2 | 1.4 ± 0.2 | 5.0 ± 1.4 | 0.88 ± 0.11 | > 200 |
| Keq (μm)f | K1/(K2+1) | 7.4 ± 8.0 | 3.0 ± 1.7 | 8.0 ± 2.1 | < 0.065 |
| Amplitudes | < 5 | < 5 | < 5 | < 5 |
a Relative to the Trp fluorescence level of the corresponding DNA-free RecQ-nucleotide complex.
b Values in parentheses indicate the extent of nucleotide-induced quench in the absence of DNA.
c Determined at [DxSO4] = 0.1 mg/ml.
d Values extrapolated to zero heparin concentration (cf. Fig. 2D).
e Value for ATPγS: 3.3 ± 0.2 s−1.
f Overall equilibrium constant of DNA binding, defined as Keq = ([RecQ(.N)]eq × [DNA]eq)/([RecQ†.(N.)DNA]eq + [RecQ††.(N.)DNA]eq) (cf. Fig. 1G).