SEQUENCE SPECIFIC DNA PROTEIN INTERACTIONS

我们的长期目标是使用限制性内切酶作为模型， 了解决定特异性的结构和能量因素 DNA-蛋白质相互作用。 我们以前的工作与EcoRI，BamHI和 EcoRV内切核酸酶已经建立了严格的热力学和动力学 的自由能变化的定量理解的基础， 蛋白质-DNA相互作用，包括直接蛋白质的贡献- 碱和蛋白质-磷酸盐接触以及扭曲 DNA. 在下一个项目期间，我们提出以下相互关联的建议 目的： 1. 为了确定和剖析熵和熵的贡献， “典型”蛋白质-DNA复合物的形成，使用滴定- 量热法、范特霍夫法和热应力法。 把这个联合收割机 使用结构扰动方法进行分析以确定侧翼如何- 序列或位点内构象决定因素影响 构象和振动熵变（Δ Δ S（o）conf和 Δ Δ（o）vib），并确定 “适应性”复合物形成的位点相差一个碱基对， （“星星”位点）和含有干扰碱基- 磷酸盐网络 2. 为了确定特定序列上下文（即，外 识别位点）影响三个位点的特异性相互作用 限制性内切核酸酶（EcoRI，BamHI，EcoRV）扭曲其DNA 以不同的方式定位，并具有不同的磷酸盐接触模式 紧邻识别位点的侧翼。 3. 为了完成我们系统的结构特征研究， 控制DNA能量贡献的DNA识别位点 DNA相互作用的影响，并扩大 本分析以BamH Ⅰ内切酶为基础。 4. 为了使用现有的EcoRI内切酶的“混杂”突变体， 确定核酸内切酶-DNA界面和能量学的 相互作用可以通过引入新的有利的 相互作用或消除不利的相互作用。

Our long-term objective is to use restriction endonucleases as models to understand the structural and energetic factors that determine specificity in DNA-protein interactions. Our previous work with EcoRI, BamHI and EcoRV endonucleases has established a rigorous thermodynamic and kinetic basis for a quantitative understanding of the free energy changes in protein-DNA interactions, including the contributions of direct protein- base and protein-phosphate contacts and the energy required to distort the DNA. For the next project period, we propose the following interrelated aims: 1. To determine and dissect the entropic and enthalpic contributions to the formation of "canonical" protein-DNA complexes, using titration- calorimetric, van't Hoff and osmotic-stress methods. To combine this analysis with structure-perturbation methods to determine how flanking- sequence or intra-site conformational determinants affect the conformational and vibrational entropy changes (DeltaDeltaS(o) conf and DeltaDelta(o) vib) and to determine the thermodynamic characteristics of the "adaptive" complexes formed with sites differing by one base-pair ("star" sites) and with sites containing base-analogs that perturb base- phosphate networks. 2. To determine how particular sequence contexts (i.e., outside a recognition site) influence site-specific interactions of three restriction endonucleases (EcoRI, BamHI, EcoRV) that distort their DNA sites in different ways and have different patterns of phosphate contacts immediately flanking the recognition site. 3. To complete our systematic study of the structural features within the DNA recognition site that govern the energetic contribution of DNA distortability to the EcoRI endonuclease-DNA interaction, and to extend this analysis to BamHI endonuclease. 4. To use existing "promiscuous" mutants of EcoRI endonuclease to determine how the endonuclease-DNA interface and the energetics of the interaction may be modified by the introduction of new favorable interactions or the elimination of unfavorable interactions.