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Nucleic Acids Research 2004 32(14):4166-4174; doi:10.1093/nar/gkh762
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Published online 9 August 2004

Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved

DNA communications by Type III restriction endonucleases—confirmation of 1D translocation over 3D looping

Luke J. Peakman and Mark D. Szczelkun*

DNA–Protein Interactions Group, 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 7439; Fax: +44 117 928 8274; Email: mark.szczelkun{at}bristol.ac.uk
Present address: L. J. Peakman, Dairy Crest Limited, Station Yard, Totnes, Devon TQ9 5JP, UK

Received June 1, 2004; Revised June 22, 2004; Accepted July 25, 2004

DNA cleavage by Type III restriction enzymes is governed strictly by the relative arrangement of recognition sites on a DNA substrate—endonuclease activity is usually only triggered by sequences in head-to-head orientation. Tens to thousands of base pairs can separate these sites. Long distance communication over such distances could occur by either one-dimensional (1D) DNA translocation or 3D DNA looping. To distinguish between these alternatives, we analysed the activity of EcoPI and EcoP15I on DNA catenanes in which the recognition sites were either on the same or separate rings. While substrates with a pair of sites located on the same ring were cleaved efficiently, catenanes with sites on separate rings were not cleaved. These results exclude a simple 3D DNA-looping activity. To characterize the interactions further, EcoPI was incubated with plasmids carrying two recognition sites interspersed with two 21res sites for site-specific recombination by Tn21 resolvase; inhibition of recombination would indicate the formation of stable DNA loops. No inhibition was observed, even under conditions where EcoPI translocation could also occur.


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