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Nucleic Acids Research 2004 32(11):3469-3479; doi:10.1093/nar/gkh685
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Published online 29 June 2004

Nucleic Acids Research, Vol. 32 No. 11 © Oxford University Press 2004; all rights reserved

One recognition sequence, seven restriction enzymes, five reaction mechanisms

Darren M. Gowers*, Stuart R.W. Bellamy 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: +44 117 954 6873; Fax: +44 117 928 8274; Email: darren.gowers{at}bristol.ac.uk

Received May 13, 2004; Revised June 7, 2004; Accepted June 16, 2004

The diversity of reaction mechanisms employed by Type II restriction enzymes was investigated by analysing the reactions of seven endonucleases at the same DNA sequence. NarI, KasI, Mly113I, SfoI, EgeI, EheI and BbeI cleave DNA at several different positions in the sequence 5'-GGCGCC-3'. Their reactions on plasmids with one or two copies of this sequence revealed five distinct mechanisms. These differ in terms of the number of sites the enzyme binds, and the number of phosphodiester bonds cleaved per turnover. NarI binds two sites, but cleaves only one bond per DNA-binding event. KasI also cuts only one bond per turnover but acts at individual sites, preferring intact to nicked sites. Mly113I cuts both strands of its recognition sites, but shows full activity only when bound to two sites, which are then cleaved concertedly. SfoI, EgeI and EheI cut both strands at individual sites, in the manner historically considered as normal for Type II enzymes. Finally, BbeI displays an absolute requirement for two sites in close physical proximity, which are cleaved concertedly. The range of reaction mechanisms for restriction enzymes is thus larger than commonly imagined, as is the number of enzymes needing two recognition sites.


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