Nucleic Acids Research Advance Access originally published online on September 8, 2009
Nucleic Acids Research 2009 37(20):6871-6880; doi:10.1093/nar/gkp726
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Nucleic Acids Research, 2009, Vol. 37, No. 20 6871-6880
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display
1Department of Immunology, University of Washington, Seattle WA 98195, 2Center for Immunity and Immunotherapies, Seattle WA 98101, 3Molecular and Cellular Biology, University of Washington, Seattle WA 98195 and 4Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle WA 98101, USA
*To whom correspondence should be addressed. Tel: +1 206 987 7314; Fax: +1 206 987 7310; Email: andrewms{at}u.washington.edu
Received June 3, 2009. Revised August 13, 2009. Accepted August 16, 2009.
Experimental analysis and manipulation of protein–DNA interactions pose unique biophysical challenges arising from the structural and chemical homogeneity of DNA polymers. We report the use of yeast surface display for analytical and selection-based applications for the interaction between a LAGLIDADG homing endonuclease and its DNA target. Quantitative flow cytometry using oligonucleotide substrates facilitated a complete profiling of specificity, both for DNA-binding and catalysis, with single base pair resolution. These analyses revealed a comprehensive segregation of binding specificity and affinity to one half of the pseudo-dimeric interaction, while the entire interface contributed specificity at the level of catalysis. A single round of targeted mutagenesis with tandem affinity and catalytic selection steps provided mechanistic insights to the origins of binding and catalytic specificity. These methods represent a dynamic new approach for interrogating specificity in protein–DNA interactions.