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Nucleic Acids Research Advance Access originally published online on October 18, 2007
Nucleic Acids Research 2007 35(21):7209-7221; doi:10.1093/nar/gkm867
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Nucleic Acids Research, 2007, Vol. 35, No. 21 7209-7221
© 2007
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

Thermodynamics of DNA target site recognition by homing endonucleases

Jennifer H. Eastberg1,2, Audrey McConnell Smith1, Lei Zhao1,3, Justin Ashworth3, Betty W. Shen1 and Barry L. Stoddard1,*

1Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025 Seattle, WA 98109, 2Graduate Program in Molecular and Cellular Biology and 3Graduate Program in Molecular Biophysics, Structure and Design, University of Washington, Seattle, WA 98195, USA

*To whom correspondence should be addressed. Tel: 1 206 667 4031; Fax: 1 206 667 6877; Email: bstoddar{at}fhcrc.org

Received August 23, 2007. Revised September 11, 2007. Accepted September 24, 2007.

The thermodynamic profiles of target site recognition have been surveyed for homing endonucleases from various structural families. Similar to DNA-binding proteins that recognize shorter target sites, homing endonucleases display a narrow range of binding free energies and affinities, mediated by structural interactions that balance the magnitude of enthalpic and entropic forces. While the balance of {Delta}H and T{Delta}S are not strongly correlated with the overall extent of DNA bending, unfavorable {Delta}Hbinding is associated with unstacking of individual base steps in the target site. The effects of deleterious basepair substitutions in the optimal target sites of two LAGLIDADG homing endonucleases, and the subsequent effect of redesigning one of those endonucleases to accommodate that DNA sequence change, were also measured. The substitution of base-specific hydrogen bonds in a wild-type endonuclease/DNA complex with hydrophobic van der Waals contacts in a redesigned complex reduced the ability to discriminate between sites, due to nonspecific {Delta}Sbinding.


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