Specificity from the synapsis of DNA elements by the SfiI endonuclease

The synapsis of DNA sites is a prerequisite for the reactions of many proteins that act at specific DNA sequences. The requirement for synapsis was investigated by analysing the reactions of SfiI, a tetrameric restriction enzyme that cleaves DNA only after Interacting with two recognition sites. In the presence of Mg2+, oligonucleotide duplexes with the cognate recognition sequence were cleaved rapidly, with cooperative kinetics, while non-cognate duplexes were not cleaved. In the absence of Mg2+, the primary complex formed by SfiI with cognate DNA contained two duplexes synapsed by the tetramer: a secondary complex containing one duplex was seen only at elevated SfiI concentrations. In contrast, the principal complex with non-cognate DNA contained one duplex bound to SfiI. Pairs of non-cognate duplexes, or one cognate and one non-cognate duplex, generally failed to form synaptic complexes. On adding Mg2+ to complexes with cognate DNA, cleavage occurred much more rapidly in the synaptic complex than in the secondary complex. DNA synapsis thus acts to enhance the specificity of SfiI for its recognition sequence, by demanding two cognate sites for a catalytically active complex and by excluding non-cognate sites from the synaptic complex. (C) 1999 Academic Press.