Restriction Endonuclease Structure and Function

描述(由申请人提供)：序列特异性的组合要素 而催化化学是发展化疗药物的核心问题。 以特定的核酸序列和结构为靶标。限制 以PvuII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 这种水解性的各个方面仍未确定。这个项目雇佣了一名 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Filter和 DNA结合的荧光分析和荧光动力学分析 停流技术。二)亲核试剂活化的测试理论 光谱方法和动力学同位素效应，并利用这些数据进行修正 建议的机制。3)探讨构象和运动在PvuII中的作用 利用多维核磁共振研究底物专一性和STAR活性 适用于较大蛋白质的光谱技术。获取的信息 从这些实验中将确定酶之间的关系 结构，所需的金属离子，以及衬底以及澄清 限制性内切酶活性的工作模式。了解结构和 这种特定的自然形式的水解性催化的功能方面是 最终设计特定序列剪刀的可行策略，它可以 针对与癌症和艾滋病相关的特定基因。

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.