DNA excision by the Sfi I restriction endonuclease
- PMID: 9698558
- DOI: 10.1006/jmbi.1998.1966
DNA excision by the Sfi I restriction endonuclease
Abstract
A mechanism for the precise excision of DNA between two target sites was elucidated by analysing the individual steps during the reactions of the SfiI endonuclease on a plasmid with two SfiI sites. Previous studies had indicated that SfiI is a tetrameric protein that binds to two copies of its recognition site before cleaving both sites in both strands. In this study, the concerted cleavage of four phosphodiester bonds was shown to arise from four consecutive reactions that had similar values for their intrinsic rate constants. Each reaction is presumably mediated by one of the four active sites in the tetramer and all four 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 dissociation of the enzyme-substrate complex at elevated temperatures. At the physiological temperature for this enzyme, all four bonds were cleaved within one minute but the subsequent dissociation of the enzyme-product complex, liberating the excised segment of DNA, took about one hour. The tetrameric structure for SfiI was confirmed by equilibrium centrifugation.
Copyright 1998 Academic Press Limited.
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