High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.

Publication Type:

Journal Article

Source:

Nucleic acids research, Volume 37, Issue 20, p.6871-80 (2009)

Keywords:

2009, Basic Sciences Division, Binding Sites, CATALYSIS, Deoxyribonucleases, Type II Site-Specific, DNA, DNA Restriction Enzymes, Endonucleases, Flow Cytometry Core Facility, Models, Molecular, Saccharomyces cerevisiae, Substrate Specificity

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.