DNA EXCISION BY THE SFII RESTRICTION-ENDONUCLEASE

A mechanism for the precise excision of DNA between two target sites w as elucidated by analysing the individual steps during the reactions o f the SfiI endonuclease on a plasmid with two SfiI sites. Previous stu dies had indicated that SfiI is a tetrameric protein that binds to two copies of its recognition site before cleaving both sites in both str ands. Ln this study, the concerted cleavage of four phosphodiester bon ds was shown to arise from four consecutive reactions that had similar values for their intrinsic rate constants. Each reaction is presumabl y mediated by one of the four active sites in the tetramer and all fou r were generally completed within the life-time of the complex between the protein and two recognition sites, though products cleaved in one or two phosphodiester bonds were also detected following premature di ssociation of the enzyme-substrate complex at elevated temperatures. A t the physiological temperature for this enzyme, all four bonds were c leaved within one minute but the subsequent dissociation of the enzyme -product complex, liberating the excised segment of DNA, took about on e hour. The tetrameric structure for SfiI was confirmed by equilibrium centrifugation. (C) 1998 Academic Press.