COOPERATIVE BINDING-PROPERTIES OF RESTRICTION-ENDONUCLEASE ECORII WITH DNA RECOGNITION SITES

EcoRII is a member of the expanding group of type IIe restriction endo nucleases that share the distinguishing feature of requiring cooperati vity between two recognition sites in their substrate DNA. To determin e the stoichiometry of the active DNA-enzyme complex and the mode of c ooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction wa s observed at dimer/site ratios of 0.25 anti 0.5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also co rresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that one EcoRII dimer is sufficient to interact cooperativ ely with two DNA recognition sites. A Lac repressor ''barrier'' bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII s ites is achieved by bending or looping of the intervening DNA stretch, Comparative cleavage of linear substrates with differently spaced int eracting sites revealed an inverse correlation between cleavage rate a nd site distance, At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.