High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.
|Title||High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.|
|Publication Type||Journal Article|
|Year of Publication||2009|
|Authors||Jarjour J, West-Foyle H, Certo MT, Hubert CG, Doyle L, Getz MM, Stoddard BL, Scharenberg AM|
|Journal||Nucleic acids research|
|Date Published||2009 Nov|
|Keywords||Binding Sites, Catalysis, Deoxyribonucleases, Type II Site-Specific, DNA, DNA Restriction Enzymes, Endonucleases, Flow Cytometry, Models, Molecular, Saccharomyces cerevisiae, Substrate Specificity|
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.
|Alternate Journal||Nucleic Acids Res.|