High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.
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.
Jarjour
Jordan
J
Department of Immunology, University of Washington, Seattle, WA 98195, USA.
West-Foyle
Hoku
H
Certo
Michael T
MT
Hubert
Christopher G
CG
Doyle
Lindsey
L
Getz
Melissa M
MM
Stoddard
Barry L
BL
Scharenberg
Andrew M
AM
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
D004247:DNA; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific