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Nucleic Acids Research Advance Access published online on February 17, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp046
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© 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

Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

Kelly L. Sanders, Lucy E. Catto, Stuart R. W. Bellamy 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 3312156; Fax: +44 117 3312168; Email: s.halford{at}bristol.ac.uk

Received December 17, 2008. Revised January 15, 2009. Accepted January 15, 2009.

Many restriction endonucleases are dimers that act symmetrically at palindromic DNA sequences, with each active site cutting one strand. In contrast, FokI acts asymmetrically at a non-palindromic sequence, cutting ‘top’ and ‘bottom’ strands 9 and 13 nucleotides downstream of the site. FokI is a monomeric protein with one active site and a single monomer covers the entire recognition sequence. To cut both strands, the monomer at the site recruits a second monomer from solution, but it is not yet known which DNA strand is cut by the monomer bound to the site and which by the recruited monomer. In this work, mutants of FokI were used to show that the monomer bound to the site made the distal cut in the bottom strand, whilst the recruited monomer made in parallel the proximal cut in the top strand. Procedures were also established to direct FokI activity, either preferentially to the bottom strand or exclusively to the top strand. The latter extends the range of enzymes for nicking specified strands at specific sequences, and may facilitate further applications of FokI in gene targeting.


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