Sequence Specific DNA Protein Interactions

序列特异性 DNA 蛋白质相互作用

• 批准号: 6879975
• 负责人: LINDA JEN-JACOBSON
• 金额: $ 37.7万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6879975
• 关键词: DNA; DNA binding protein; acidity /alkalinity; binding sites; chemical kinetics; computer simulation; enzyme activity; intermolecular interaction; ionic bond; microcalorimetry; model design /development; molecular dynamics; molecular energy level; molecular shape; nucleic acid sequence; physical model; protein structure function; radiotracer; restriction endonucleases; scintillation spectrometry; site directed mutagenesis; stereochemistry; thermodynamics; water

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.

描述（申请人提供）：特定碱基的蛋白质识别 双螺旋 DNA 中的序列是许多生物过程的核心， 包括遗传信息的调控和表达 位点特异性重组。该项目的长期目标是利用 限制性内切酶作为模型来了解结构和能量 决定位点特异性 DNA-蛋白质相互作用特异性的因素。 之前使用 EcoRI 和 BamHI 核酸内切酶的工作已经建立了严格的 定量理解变化的热力学和动力学基础 自由能 (deltaGdeg)、焓 (deltaHdeg) 和熵 （deltaS Degree）在蛋白质-DNA复合物的形成中。该方法旨在 将结构性和能量性的观点结合到特异性和测试中 所提出原则的一般性。现在建议确定如何 分子应变（EcoRV 复合体中的 DNA 弯曲；EcoRV 复合体中的静电排斥） BamHI 复合物）影响热力学参数（deltaH 度、deltaS 度 和 deltaC Degreep），并确定特定的序列上下文（即， DNA 识别位点之外）起到调节特异性和调整的作用 EcoRI 和 EcoRV 核酸内切酶 DNA 复合物中的分子菌株。 现有的和新设计的 EcoRI 核酸内切酶突变体将用于 确定如何引入新的有利相互作用或消除 不利的相互作用通过改变结构来放松特异性 蛋白质-DNA 界面、结合和/或裂解的热力学 系统的动力学。 BamHI 和 EcoRV 核酸内切酶将用于 评估 H20 的排除共溶质方法的定量校准 蛋白质-DNA 结合过程中的释放，并比较实验结果 与计算预测。