High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display |
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Authors: | Jordan Jarjour Hoku West-Foyle Michael T Certo Christopher G Hubert Lindsey Doyle Melissa M Getz Barry L Stoddard Andrew M Scharenberg |
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Institution: | 1.Department of Immunology, University of Washington, Seattle WA 98195, 2.Center for Immunity and Immunotherapies, Seattle WA 98101, 3.Molecular and Cellular Biology, University of Washington, Seattle WA 98195 and 4.Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle WA 98101, USA |
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Abstract: | 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. |
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