Restriction Endonuclease Structure and Function

限制性内切酶的结构和功能

• 批准号: 6745111
• 负责人: CYNTHIA M. DUPUREUR
• 金额: $ 21.41万
• 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/6745111
• 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）：结合序列特异性元素