Sequence Specific DNA Protein Interactions

性状（申请人提供）：特异性碱基的蛋白质识别 双螺旋DNA中的序列是许多生物过程的核心， 包括遗传信息的调节和表达， 位点特异性重组。该项目的长期目标是利用 限制性内切核酸酶作为模型，以了解结构和能量 决定位点特异性DNA-蛋白质相互作用特异性的因素。 先前使用EcoRI和BamHI内切核酸酶的工作已经建立了严格的 热力学和动力学基础的定量理解的变化 在自由能（deltaG度）、焓（deltaH度）和熵中 （deltaSdegree）在蛋白质-DNA复合物形成中的作用。该方法旨在 结合结构和能量的角度对特异性和测试 提出的原则的一般性。现在建议确定如何 分子应变（EcoRV复合物中的DNA弯曲; BamHI复合物）影响热力学参数（Δ H度，Δ S度 和deltaCdegreep），并确定特定序列上下文（即， 在DNA识别位点外）起调节特异性和调节 在EcoRI和EcoRV内切核酸酶的DNA复合物中的分子菌株。 现有的和新工程改造的EcoRl内切核酸酶突变体将用于 确定如何引入新的有利的相互作用或消除 不利的相互作用通过改变结构放松特异性， 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核酸内切酶将用于 排除共溶质法测定H20的定量校正 释放过程中蛋白质-DNA协会，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.