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Nucleic Acids Research Advance Access originally published online on February 14, 2008
Nucleic Acids Research 2008 36(6):2073-2081; doi:10.1093/nar/gkn051
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Nucleic Acids Research, 2008, Vol. 36, No. 6 2073-2081
© 2008 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

Dynamics and consequences of DNA looping by the FokI restriction endonuclease

Lucy E. Catto, Stuart R. W. Bellamy, Susan E. Retter and Stephen E. Halford*

The DNA-Protein Interactions Unit, Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK

*To whom correspondence should be addressed. Tel: +44 117 331 2156; Fax: +44 117 331 2168; Email: s.halford{at}bristol.ac.uk

Received January 3, 2008. Revised January 25, 2008. Accepted January 25, 2008.

Genetic events often require proteins to be activated by interacting with two DNA sites, trapping the intervening DNA in a loop. While much is known about looping equilibria, only a few studies have examined DNA-looping dynamics experimentally. The restriction enzymes that cut DNA after interacting with two recognition sites, such as FokI, can be used to exemplify looping reactions. The reaction pathway for FokI on a supercoiled DNA with two sites was dissected by fast kinetics to reveal, in turn: the initial binding of a protein monomer to each site; the protein–protein association to form the dimer, trapping the loop; the subsequent phosphodiester hydrolysis step. The DNA motion that juxtaposes the sites ought on the basis of Brownian dynamics to take ~2 ms, but loop capture by FokI took 230 ms. Hence, DNA looping by FokI is rate limited by protein association rather than DNA dynamics. The FokI endonuclease also illustrated activation by looping: it cut looped DNA 400 times faster than unlooped DNA.


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