Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.
Determination of protein-DNA binding constants and specificities from statistical analyses of single molecules: MutS-DNA interactions.
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DOI：
10.1093/nar/gki708
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发表时间：
2005
影响因子：
14.9
通讯作者：
Erie DA
中科院分区：
文献类型：
--
作者：
Yang Y;Sass LE;Du C;Hsieh P;Erie DA
原子力显微镜(AFM)是研究蛋白质-DNA复合体构象和确定蛋白质-蛋白质复合体的化学计量和亲和力的一种强有力的技术。我们将原子力显微镜的能力扩展到蛋白质-DNA结合常数和特异性的测定。蛋白质在DNA片段上的位置分布提供了特异性的直接测量，而不需要知道绝对结合常数。蛋白质在给定位置的占有率与蛋白质和DNA的浓度相结合，可以确定绝对结合常数。我们介绍了这一分析的理论基础，并通过表征MutS与包含不错配或单一错配的DNA片段的相互作用来证明其实用性。我们表明，MutS对错配的特异性比之前从批量研究中提出的要高得多，而且明显的低特异性是高亲和力与DNA末端结合的结果。这些结果解决了MutS明显的低结合特异度和预期的高修复特异度的难题。总之，通过一组AFM实验，可以确定蛋白质-DNA复合体的结合亲和力、特异性和化学计量比，以及构象性质。
Atomic force microscopy (AFM) is a powerful technique for examining the conformations of protein–DNA complexes and determining the stoichiometries and affinities of protein–protein complexes. We extend the capabilities of AFM to the determination of protein–DNA binding constants and specificities. The distribution of positions of the protein on the DNA fragments provides a direct measure of specificity and requires no knowledge of the absolute binding constants. The fractional occupancies of the protein at a given position in conjunction with the protein and DNA concentrations permit the determination of the absolute binding constants. We present the theoretical basis for this analysis and demonstrate its utility by characterizing the interaction of MutS with DNA fragments containing either no mismatch or a single mismatch. We show that MutS has significantly higher specificities for mismatches than was previously suggested from bulk studies and that the apparent low specificities are the result of high affinity binding to DNA ends. These results resolve the puzzle of the apparent low binding specificity of MutS with the expected high repair specificities. In conclusion, from a single set of AFM experiments, it is possible to determine the binding affinity, specificity and stoichiometry, as well as the conformational properties of the protein–DNA complexes.
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