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Nucleic Acids Research, 2003, Vol. 31, No. 18 5221-5228
© 2003 Oxford University Press

DNA supercoiling enables the Type IIS restriction enzyme BspMI to recognise the relative orientation of two DNA sequences

Isabel J. Kingston, Niall A. Gormley and Stephen E. Halford*

Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK

*To whom correspondence should be addressed. Tel: +44 117 928 7429; Fax: +44 117 928 8274; Email: s.halford{at}bristol.ac.uk
Present address:
Niall A. Gormley, Solexa Ltd, Chesterford Research Park, Essex CB10 1XL, UK

Many proteins can sense the relative orientations of two sequences at distant locations in DNA: some require sites in inverted (head-to-head) orientation, others in repeat (head-to-tail) orientation. Like many restriction enzymes, the BspMI endonuclease binds two copies of its target site before cleaving DNA. Its target is an asymmetric sequence so two sites in repeat orientation differ from sites in inverted orientation. When tested against supercoiled plasmids with two sites 700 bp apart in either repeated or inverted orientations, BspMI had a higher affinity for the plasmid with repeated sites than the plasmid with inverted sites. In contrast, on linear DNA or on supercoiled DNA with sites 1605 bp apart, BspMI interacted equally with repeated or inverted sites. The ability of BspMI to detect the relative orientation of two DNA sequences thus depends on both the topology and the length of the intervening DNA. Supercoiling may restrain the juxtaposition of sites 700 bp apart to a particular alignment across the superhelical axis, but the juxtaposition of sites in linear DNA or far apart in supercoiled DNA may occur without restraint. BspMI can therefore act as a sensor of the conformational dynamics of supercoiled DNA.


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