Table 1. Specific activities of wild-type and mutant Hex1-N2 proteins for double-stranded (D.S.) blunt end and 5′-flap DNA substrates.
| Specific activity (D.S.) (pmol min–1 mg–1) | Fold reduction | Specific activity (5′-flap) (pmol min–1 mg–1) | Fold reduction | |
|---|---|---|---|---|
| Hex1-N2 | (0.97 ± 0.3) × 105 | (0.89 ± 0.2) × 105 | ||
| D78A | (0.47 ± 0.2) × 103 | 206 | (0.86 × 0.1) × 102 | 1035 |
| D173A | (1.74 ± 0.4) × 103 | 56 | (1.33 ± 0.1) × 102 | 669 |
| D225A | (1.00 ± 0.1) × 103 | 97 | (2.34 ± 0.3) × 102 | 380 |
The averages and standard deviations of three independent nuclease reactions are shown, as well as the fold reduction in activity compared to wild-type Hex1-N2. The oligonucleotides used to create the double-stranded DNA substrate were *5′-TAG AGG ATC CCC GCT AGC GGG TAC CGA GCT CGA ATT CAC TGG-3′ and 5′-CCA GTG AAT TCG AGC TCG GTA CCC GCT AGC GGG GAT CCT CTA-3′, and to create the 5′-flap DNA were *5′-ATT GGT TAT TTA CCG AGC TCG AAT TCA CTG G-3′, 5′-TAG AGG ATC CCC GCT AGC GGG-3′ and 5′-CCA GTG AAT TCG AGC TCG GTA CCC GCT AGC GGG GAT CCT CTA-3′. The asterisk indicates the oligonucleotide that was 32P-labeled. See Materials and Methods for details.