Figure 1.

The product distribution of T5 exonuclease reaction varies with pH. (A) Activity was assayed on the pseudo-Y substrate, with 10 mM MgCl₂ cofactor. Lanes marked M show the result of reactions in mixtures that contained no enzyme, and lanes 1–6 contained 4 nM wild-type enzyme in buffers at pH 5.5, 6.0, 7.0, 8.0, 9.3, and 11.3, respectively. The sizes (nucleotides) of the products are as indicated. (B) Time courses of wild-type 5′-3′ exonuclease at pH extremes. Wild-type exonuclease, at a concentration of 0.8 nM, was incubated with the labeled pseudo-Y substrate at 37°C for 2.5, 5, or 10 min at pH 5.5 (lanes 1–3) or pH 9.3 (lanes 4–6) in the presence of 10 mM MgCl₂. The products of the reaction were separated on a 7 M urea/15% acrylamide gel. Untreated substrate, lane M, and substrates incubated at 37°C for 10 min at each pH in the absence of enzyme (lanes 7 and 8) are shown. (C) Phosphoimager data from A showing the percentage of product plotted against pH for both exonucleolytic (▴) and endonucleolytic (■) cleavage. (D) Graphical representation of the time course at pH 5.5 and 9.3 from data obtained from B. Endonucleolytic product is shown by dark bars, exonucleolytic product, by light bars. Differences between C and D in the absolute levels of exo- and endonuclease activity reflect the concentration of the enzyme used. In C all of the original substrate has been degraded.