DNA CLEAVAGE AT 2 RECOGNITION SITES BY THE SFII RESTRICTION-ENDONUCLEASE - SALT DEPENDENCE OF CIS AND TRANS INTERACTIONS BETWEEN DISTANT DNA SITES

At low ionic strength, the SfiI restriction enzyme cleaved at similar rates both supercoiled and linear DNA with two SfiI sites and linear D NA with one SfiI site. For the substrates with two sites, the majority of the DNA was converted directly to products cut at both sites; the enzyme appears to bind to two sites before catalyzing its reactions, l ooping out the intervening DNA. At high ionic strength, linear DNA wit h one SfiI site was not cut at all linear DNA with two sites was cleav ed slowly while supercoiled DNA with two sites was cleaved rapidly, th ough only half of the DNA with two sites was cut at both sites; the DN A that had been cut at one site was not cleaved again at the remaining site. The singly cut product must therefore have been generated by a reaction incorporating both sites. All DNA cleavage reactions by SfiI thus involve the tetrameric enzyme bound to two copies of its recognit ion sequence, but weakened DNA-protein interactions at high ionic stre ngth can cause this complex to dissociate before cleaving both sites. Intramolecular interactions between distant DNA sites are generally th ought to be enhanced by supercoiling and to be more stable than interm olecular interactions. The preference of SfiI at high ionic strength f or substrates with two sites over substrates with one site and, in the former ease, for supercoiled over linear DNA, validates this view. At low ionic strength, the similar rates with the different substrates m ay be due to rate-limiting product dissociation. (C) 1995 Academic Pre ss Limited