Figure 6.

Optimisation of the PI-MgaI endonuclease activity. Aliquots of 100 ng pMgaSite DNA substrate were incubated for 60 min at 37°C with the soluble crude extract of untagged MgaPps1 under different conditions. These data represent single observations. (A) Effect of buffer composition and pH. The assays were performed at different pH values with 70 ng proteins, in 10 mM Tris–acetate (diamonds) or Tris–HCl (squares) buffers containing 10 mM MgCl₂ and 10 mM NaCl. (B) Effect of MgCl₂ concentration. The assays were performed with 50 ng proteins, in 10 mM Tris–HCl, pH 8, 10 mM NaCl and increasing concentrations of MgCl₂. (C) Effect of monovalent ions. The assays were performed with 60 ng proteins, in 10 mM Tris–HCl, pH 8, 10 mM MgCl₂ and increasing concentrations of NaCl (squares), KCl (diamonds) or NH₄OAc (triangles). (D) Effect of KCl concentration. The assays were performed in 10 mM Tris–HCl, pH 8, 10 mM MgCl₂ and increasing KCl concentrations ranging from 10 to 50 mM, in the presence of only 20 ng of the crude extract of untagged MgaPps1.