Restriction Endonuclease Structure and Function

限制性内切酶的结构和功能

• 批准号: 6594244
• 负责人: CYNTHIA M. DUPUREUR
• 金额: $ 20.58万
• 依托单位: UNIVERSITY OF MISSOURI-ST. LOUIS
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6594244
• 关键词: DNA binding protein; atomic absorption spectrometry; conformation; divalent cations; divalent metal; enzyme activity; enzyme structure; fluorescence spectrometry; hydrolysis; nuclear magnetic resonance spectroscopy; protein structure function; relaxation spectrometry; restriction endonucleases; stop flow technique

DESCRIPTION (provided by applicant): Combining elements of sequence-specificity and catalytic chemistry is the central problem in developing chemotherapeutics which target specific nucleic acid sequences and structures. Restriction enzymes, for which PvuII endonuclease serves as a model, are the simplest biological agents of site-specific nucleic acid chemistry, yet many mechanistic aspects of this hydrolytic activity remain undefined. This project employs a unique combination of techniques to: i) Dissect the roles of metal ions in DNA binding and hydrolysis. These goals will be accomplished with filter and fluorescence assays of DNA binding and kinetic assays using fluorescence stopped-flow techniques. ii) Test theories of nucleophile activation using spectroscopic methods and kinetic isotope effects and use this data to revise proposed mechanisms. iii) Probe the roles of conformation and motion in PvuII substrate specificity and star activity utilizing multidimensional NMR spectroscopic techniques applicable to larger proteins. Information obtained from these experiments will identify the relationships between the enzyme structure, the required metal ions, and the substrate as well as clarify working models of restriction enzyme activity. Understanding the structural and functional aspects of this natural form of specific, hydrolytic catalysis is a viable strategy to the eventual design of sequence-specific scissors which can target specific genes linked to cancer and AIDS.

描述（由申请人提供）：组合序列特异性的元素 催化化学是开发化疗药物的核心问题 它针对特定的核酸序列和结构。限制 以 PvuII 核酸内切酶为模型的酶是最简单的 位点特异性核酸化学的生物制剂，但许多机制 这种水解活性的各个方面仍不清楚。该项目采用了 独特的技术组合：i) 剖析金属离子在 DNA 中的作用 结合和水解。这些目标将通过过滤器和 DNA 结合的荧光测定和使用荧光的动力学测定 停流技术。 ii) 使用以下方法测试亲核试剂活化理论 光谱方法和动力学同位素效应，并使用该数据进行修正 拟议的机制。 iii) 探讨 PvuII 中构象和运动的作用 利用多维 NMR 确定底物特异性和星号活性 适用于较大蛋白质的光谱技术。获得的信息 从这些实验中将确定酶之间的关系 结构、所需的金属离子和底物以及澄清 限制酶活性的工作模型。了解结构和 这种特定的自然形式的水解催化的功能方面是 最终设计序列特异性剪刀的可行策略 靶向与癌症和艾滋病相关的特定基因。