Published online 23 March 2005
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On the DNA cleavage mechanism of Type I restriction enzymes
Biozentrum of the University of Basel, Division of Molecular Microbiology Klingelbergstrasse 50-70, CH-4056 Basel, Switzerland 1Institute of Molecular Cancer Research, University of Zürich August Forel Strasse 7, CH-8008 Zürich, Switzerland
*To whom correspondence should be addressed. Tel: +41 0 61 2672120; Fax: +41 0 61 2672118; Email: thomas-a.bickle{at}unibas.ch
Received January 5, 2005. Revised February 3, 2005. Accepted March 6, 2005.
Although the DNA cleavage mechanism of Type I restriction–modification enzymes has been extensively studied, the mode of cleavage remains elusive. In this work, DNA ends produced by EcoKI, EcoAI and EcoR124I, members of the Type IA, IB and IC families, respectively, have been characterized by cloning and sequencing restriction products from the reactions with a plasmid DNA substrate containing a single recognition site for each enzyme. Here, we show that all three enzymes cut this substrate randomly with no preference for a particular base composition surrounding the cleavage site, producing both 5'- and 3'-overhangs of varying lengths. EcoAI preferentially generated 3'-overhangs of 2–3 nt, whereas EcoKI and EcoR124I displayed some preference for the formation of 5'-overhangs of a length of 
6–7 and 3–5 nt, respectively. A mutant EcoAI endonuclease assembled from wild-type and nuclease-deficient restriction subunits generated a high proportion of nicked circular DNA, whereas the wild-type enzyme catalyzed efficient cleavage of both DNA strands. We conclude that Type I restriction enzymes require two restriction subunits to introduce DNA double-strand breaks, each providing one catalytic center for phosphodiester bond hydrolysis. Possible models for DNA cleavage are discussed.
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