Sequence Specific DNA Protein Interactions

描述(申请人提供)：特定碱基的蛋白质识别 双螺旋DNA中的序列位于许多生物过程的核心， 包括遗传信息的调节和表达，以及 特定部位重组。该项目的长期目标是使用 限制性内切酶作为理解结构和能量的模型 决定特定部位DNA-蛋白质相互作用的特异性的因素。 之前对EcoRI和BamHI内切酶的研究已经建立了严格的 定量理解变化的热力学和动力学基础 在自由能(deltaG度)、焓(deltaH度)和熵中 (增量)形成蛋白质-DNA复合体。这种方法的目的是 将结构和能量观点统一到专一性上，并测试 所援引原则的概括性。现在有人提议确定如何 分子应变(EcoRV复合体中的DNA弯曲； BamHI复合体)影响热力学参数(DeltaH度、DeltaS度 和DeltaCderep)，并确定特定的序列上下文(即， 在DNA识别位点之外)起调节特异性和调节 EcoRI和EcoRV内切酶DNA复合体中的分子菌株。 现有的和新设计的EcoRl内切酶突变体将用于 确定如何引入新的有利互动或消除 不利相互作用的影响通过改变 蛋白质-DNA界面、结合和/或切割的热力学 系统的动力学。BamHI和EcoRV内切酶将用于 测定H20的排除共溶液法的定量标定 蛋白质-DNA结合过程中的释放，并比较实验结果 通过计算预测。

DESCRIPTION (provided by applicant): Protein recognition of specific base sequences in double-helical DNA lies at the heart of many biological processes, including the regulation and expression of genetic information and site-specific recombination. The long-term objective of this project is to use restriction endonucleases as models to understand the structural and energetic factors that determine specificity in site-specific DNA-protein interactions. Previous work with EcoRI and BamHI endonucleases has established a rigorous thermodynamic and kinetic basis for a quantitative understanding of the changes in free energy (deltaGdegree), enthalpy (deltaHdegree) and entropy (deltaSdegree) in formation of protein-DNA complexes. The approach is aimed at uniting structural and energetic perspectives on specificity and at testing the generality of the principles adduced. It is now proposed to determine how molecular strain (DNA bending in the EcoRV complex; electrostatic repulsion in the BamHI complex) affects thermodynamic parameters (deltaHdegree, deltaSdegree and deltaCdegreep), and to determine how particular sequence contexts (i.e., outside a DNA recognition site) act to modulate specificity and to adjust molecular strain in the DNA complexes of EcoRI and EcoRV endonucleases. Existing and newly-engineered mutants of EcoRl endonuclease will be used to determine how the introduction of new favorable interactions or the elimination of unfavorable interactions relaxes specificity by modifying the structure of the protein-DNA interface, the thermodynamics of binding and/or the cleavage kinetics of the system. The BamHI and EcoRV endonucleases will be used for quantitative calibration of the excluded-cosolute method of evaluating H20 release during protein-DNA association, and to compare experimental results with computational predictions.