Figure 3.

Structure-specific cleavage of Ps-Y (A) and 5OVH substrates (B) by wild-type (WT) and mutant enzymes. Enzymes, at ≈1 nM wild-type and 10 nM for the mutants, were incubated at 37°C with substrate DNA [strand F1 labeled in Ps-Y (A) or B1 in 5OVH (B), 150 pM] in the presence of 10 mM MgCl₂ for 0.5, 2, 5, 10, and 20 min (indicated by the filled triangles). Reaction products were separated by denaturing PAGE and visualized by autoradiography. Controls (0) that lacked enzyme but contained all other reactants were incubated for 30 min. Single nucleotide ladders (M) were produced by digestion of labeled F1 (A) or B1 (B) oligonucleotide with snake venom phosphodiesterase. Mutant exonucleases R33A, R172A, K241A, and K215A gave very similar results to those above (data not shown).