Restriction Endonuclease Structure and Function

描述（由申请人提供）：组合序列特异性元件 催化化学是发展化学治疗的中心问题 其靶向特定的核酸序列和结构。限制 以PvuII内切核酸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 这种水解活性的方面仍然不确定。该项目采用了 独特的技术组合，以：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）亲核试剂活化的测试理论， 光谱方法和动力学同位素效应，并利用这些数据来修正 提出的机制。iii）探索PvuII中构象和运动的作用 利用多维NMR底物特异性和星星活性 光谱技术适用于较大的蛋白质。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，和基板以及澄清 限制性内切酶活性的工作模型了解结构和 这种天然形式的功能方面的具体，水解催化是一个 最终设计序列特异性剪刀的可行策略， 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.