title: High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.
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.
authors.affiliation: Department of Immunology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.forename: Hoku
authors.initials: H
authors.lastname: West-Foyle
authors.forename: Michael T
authors.initials: MT
authors.lastname: Certo
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Hubert
authors.forename: Lindsey
authors.initials: L
authors.lastname: Doyle
authors.forename: Melissa M
authors.initials: MM
authors.lastname: Getz
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004720:Endonucleases / Q000378:metabolism; D005434:Flow Cytometry; D008958:Models, Molecular; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004247:DNA; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific