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Nucleic Acids Research Advance Access originally published online on February 17, 2009
Nucleic Acids Research 2009 37(6):2053-2063; doi:10.1093/nar/gkp042
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Nucleic Acids Research, 2009, Vol. 37, No. 6 2053-2063
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Atomic force microscopy of the EcoKI Type I DNA restriction enzyme bound to DNA shows enzyme dimerization and DNA looping

Kelly J. Neaves1, Laurie P. Cooper2, John H. White2, Stewart M. Carnally1, David T. F. Dryden2, J. Michael Edwardson1 and Robert M. Henderson1,*

1Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD and 2School of Chemistry, The King's Buildings, The University of Edinburgh, Edinburgh EH9 3JJ, UK

*To whom correspondence should be addressed. Tel: +44 1223 334 053; Fax: +44 1223 334 100; Email: rmh1003{at}cam.ac.uk

Received October 29, 2008. Revised January 2, 2009. Accepted January 14, 2009.

Atomic force microscopy (AFM) allows the study of single protein–DNA interactions such as those observed with the Type I Restriction–Modification systems. The mechanisms employed by these systems are complicated and understanding them has proved problematic. It has been known for years that these enzymes translocate DNA during the restriction reaction, but more recent AFM work suggested that the archetypal EcoKI protein went through an additional dimerization stage before the onset of translocation. The results presented here extend earlier findings confirming the dimerization. Dimerization is particularly common if the DNA molecule contains two EcoKI recognition sites. DNA loops with dimers at their apex form if the DNA is sufficiently long, and also form in the presence of ATP{gamma}S, a non-hydrolysable analogue of the ATP required for translocation, indicating that the looping is on the reaction pathway of the enzyme. Visualization of specific DNA loops in the protein–DNA constructs was achieved by improved sample preparation and analysis techniques. The reported dimerization and looping mechanism is unlikely to be exclusive to EcoKI, and offers greater insight into the detailed functioning of this and other higher order assemblies of proteins operating by bringing distant sites on DNA into close proximity via DNA looping.


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