Table 1.
Effects of mutations on RecE exonuclease and DNA-binding activities.
| Protein or mutant of RecE564 | kcat (sec−1) | Kd (nM) |
|---|---|---|
| WT (RecE564) | 18.8 ± 1.9 | 70 ± 2 |
| P658L (RecE606) | 0.42 ± 0.01 | 110 ± 20 |
| λ exonuclease | 9.3 ± 0.4 | 161 ± 2 |
| Motif I | ||
| E729A | 0.03 ± 0.08 | 57 ± 3 |
| Motif II | ||
| R744A | 6.6 ± 0.7 | 95 ± 8 |
| R746A | 0.3 ± 0.1 | 90 ± 10 |
| Motif III | ||
| K761A | 0.16 ± 0.04 | 130 ± 10 |
| Motif IV | ||
| Y778A | 0.2 ± 0.2 | 130 ± 20 |
| Q781A | 6.2 ± 0.4 | 70 ± 20 |
| Y785A | 0.4 ± 0.1 | 98 ± 9 |
| Motif V | ||
| H652A | 0.02 ± 0.04 | 130 ± 10 |
| E657A | 0.8 ± 0.1 | 90 ± 10 |
| P658L | 14.4 ± 0.1 | 80 ± 10 |
| Motif VI | ||
| Y608A | 0.6 ± 0.1 | 160 ± 10 |
| H609A | 2.1 ± 0.1 | 50 ± 3 |
| S615A | 14.6 ± 0.1 | 100 ± 4 |
| S617A | 4.5 ± 0.3 | 150 ± 20 |
| Disordered loop (residues 665–698) | ||
| F661A | 14.7 ± 0.6 | 118 ± 8 |
| F665A | 16.4 ± 0.3 | 200 ± 20 |
| P669A | 23.2 ± 1.3 | 109 ± 8 |
| T675A | 23.6 ± 0.9 | 650 ± 30 |
| Central channel | ||
| K643A | 22.3 ± 0.8 | 210 ± 6 |
| K704A | 24.6 ± 2.2 | 330 ± 50 |
| R768A | 25.5 ± 0.6 | 310 ± 30 |
| C-terminal plug | ||
| R858A | 28.3 ± 1.9 | 77 ± 5 |
| W859A | 27.8 ± 2.0 | 350 ± 20 |
| Δ858–866 | 6.4 ± 0.5 | 660 ± 80 |
Values of kcat in nucleotides per second per DNA end were measured for each purified protein by the fluorescence-based assay as demonstrated in Figure 7a,b and described in Experimental Procedures. Values of Kd (nM) were determined for each protein by fluorescence anisotropy as demonstrated in Figure 7c and described in Experimental Procedures. All standard deviations are based on measurements from at least three independent experiments.