Jarjour Jordan, West-Foyle Hoku, Certo Michael T, Hubert Christopher G, Doyle Lindsey, Getz Melissa M, Stoddard Barry L, Scharenberg Andrew M
Department of Immunology, University of Washington, Seattle, WA 98195, USA.
Nucleic Acids Res. 2009 Nov;37(20):6871-80. doi: 10.1093/nar/gkp726. Epub 2009 Sep 8.
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.
对蛋白质 - DNA 相互作用进行实验分析和操作面临着独特的生物物理挑战，这些挑战源于 DNA 聚合物的结构和化学均一性。我们报道了利用酵母表面展示技术进行基于分析和筛选的应用，用于 LAGLIDADG 归巢内切酶与其 DNA 靶点之间的相互作用。使用寡核苷酸底物的定量流式细胞术有助于以单碱基对分辨率对 DNA 结合和催化的特异性进行全面分析。这些分析揭示了结合特异性和亲和力在假二聚体相互作用的一半区域存在全面分离，而整个界面在催化水平上贡献特异性。通过一轮具有串联亲和力和催化筛选步骤的靶向诱变，为结合和催化特异性的起源提供了机制见解。这些方法代表了一种用于探究蛋白质 - DNA 相互作用特异性的动态新方法。
