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来 了解决定特异性的结构和能量因素 在DNA和蛋白质的相互作用中。我们之前与EcoRI、BamHI和 EcoRV内切酶已经建立了严格的热力学和动力学 对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- 碱基和蛋白质-磷酸盐的接触以及扭曲 DNA对于下一个项目期，我们提出了以下相互关联的 目标： 1.确定和剖析对……的熵和焓贡献 用滴定的方法形成“正则”蛋白质-DNA复合体。 量热法、范特霍夫法和渗透压应激法。要将这一点结合起来 用结构摄动法进行分析以确定侧翼如何 序列或站点内构象决定因素影响 构象和振动熵变化(DeltaDeltaS(O)conf和 DeltaDelta(O)VIB)，并确定其热力学特性 由一个碱基对不同的位点形成的“适应性”复合体 以及含有扰乱碱基的碱基类似物的位点- 磷酸盐网络。 2.确定特定的序列上下文(即，在 识别位置)影响三个位置特定的相互作用 扭曲DNA的限制性内切酶(EcoRI、BamHI、EcoRV) 部位有不同的方式，并有不同的磷酸盐接触模式 立即在辨认地点的侧翼。 3.完成我们对内部结构特征的系统研究 支配DNA能量贡献的DNA识别位点 对EcoRI内切酶-DNA相互作用的扭曲，并扩展 这是对BamHI内切酶的分析。 4.利用已有的EcoRI内切酶“混杂”突变体 确定内切酶-DNA界面和细胞的能量学 互动可能会通过引入新的有利条件来修改 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.