Figure 1.

Salt and pH dependence of Hs-RNase H1. The action of Hs-RNase H1 on the 21 bp hybrid substrate was measured as a function of KCl concentration (panels A,B) or pH (panels C,D) in reactions performed at 30°C in buffer containing 10 mM MgCl₂, and 10 mM DTT (see Materials and Methods). Both analyses used 30 nM Hs-RNase H1 and 100 nM 5′-³²P-labeled (preformed) hybrid. For the KCl concentration dependence experiment, 20 mM Tris-HCl (pH 7.5) was used as buffer. In panel A, lanes 2–9 show reactions at KCl concentrations of 25, 50, 100, 150, 200, 300, 400 and 600 mM, respectively. Lane 1 is a control reaction that lacked Mg²⁺. Panel B graphically displays the fraction of substrate cleaved as a function of KCl concentration. The maximum error is shown for each point. C. pH dependence. Lanes 2–14 show reactions at pH values of 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, and 11.5, respectively. The KCl concentration was 150 mM. Tris buffer was used at each pH value. Here, the use of Tris rather than HEPES for pH titration at fixed Mg²⁺ concentration allowed the 5.5–11.5 pH range to be achieved. Panel D displays the fraction of substrate cleaved as a function of pH. Experiments were carried out in duplicate, and the average values are shown with the associated maximum error.