High-resolution profiling of homing endonuclease binding and catalytic specificity using yeast surface display.
Nucleic Acids Res
; 37(20): 6871-80, 2009 Nov.
Article
em En
| MEDLINE
| ID: mdl-19740766
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Desoxirribonucleases de Sítio Específico do Tipo II
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Estados Unidos