Specificity from the synapsis of DNA elements by the Sfi I 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 Sfi I, 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 Sfi I with cognate DNA contained two duplexes synapsed by the tetramer: a secondary complex containing one duplex was seen only at elevated Sfi I concentrations. In contrast, the principal complex with non-cognate DNA contained one duplex bound to Sfi I. 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 Sfi I for its recognition sequence, by demanding two cognate sites for a catalytically active complex and by excluding non-cognate sites from the synaptic complex.
Embleton
M L
ML
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol, BS8 1TD, UK.
Williams
S A
SA
Watson
M A
MA
Halford
S E
SE
D002384:Catalysis; D004247:DNA / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011485:Protein Binding; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific