The influence of divalent metal ions on DNA binding and cleavage by the restriction enzyme PvuII endonuclease

Divalent metal ions can play an important role in the interactions between nucleic acids and proteins. Here, PvuII endonuclease is developed as a model system to study DNA binding and catalysis as a function of divalent metal ions. A novel approach of equilibrium binding and kinetics of binding and cleavage is used to understand the influence of divalent metal ions on DNA recognition and catalysis. In the presence of calcium, a divalent metal ion that does not support catalysis, the interaction between the specific recognition site and PvuII endonuclease has a K_d of 53 ± 10 pM (pH 7.5, 100 mM NaCl, 10 MM CaCl₂). A 6000 fold reduction in the dissociation constant is seen when metal ions are absent. Specific DNA binding interactions exhibit an unusual shallow pH dependence. Most protein-DNA interactions have a more pronounced pH effect. Nonspecific DNA binding is independent of divalent metal ions; exhibiting a K_d near 200 nM (pH 7.5, 100 mM NaCl).; Kinetic methods were developed to understand how many metal ions are involved with both DNA binding and catalysis. This work presents the first comprehensive kinetic study of a restriction enzyme to show that more than one metal ion influences both the DNA cleavage and association rate constants. Using Ca(II) to prevent turnover, the enzyme-DNA association rate constant is metal ion concentration dependent, exhibiting a 100 fold increase from metal-free experiments to those done in the presence of 10 mM Ca(II). The association rate constant exhibited cooperative binding of at least four metal ions per PvuII endonuclease dimer. The dissociation rate constant showed a shallow Ca(II) concentration dependence. This provides new evidence that the metal ion influence on the DNA dissociation constant is due to alterations in the association rate constant. Hill analysis of the cleavage rate constant as a function of Mg(II) concentration, indicates that at least three metal ions per endonuclease dimer are involved in DNA hydrolysis. Combining the knowledge gained from equilibrium and kinetic studies provides unique insights on how metal ions influence DNA binding and catalysis for restriction enzymes.