MECHANISM OF RESTRICTION ENDONUCLEASE ACTION

限制性核酸内切酶作用机制

• 批准号: 3299467
• 负责人: FRANCIS BARANY
• 金额: $ 19.98万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1994-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3299467
• 关键词: DNA; DNA binding protein; allosteric site; chemical structure function; endonuclease; enzyme mechanism; enzyme structure; gel electrophoresis; genetic manipulation; hydrogen bond; messenger RNA; molecular cloning; nucleic acid sequence; oligonucleotides; plasmids; protein sequence; site directed mutagenesis

The long term goal of this project is to understand the basis of sequence-specific recognition of DNA by proteins. DNA recognition governs many metabolic activities including gene activation and repression, site-specific recombination and initiation of DNA replication. Understanding how these proteins work will provide a framework for the rational design of new protein specificities that may have medical and pharmacological applications. The particular protein of study is the thermophilic restriction endonuclease TagI. A model has been proposed for TagI recognition of its cognate sequence. Further refinement of this model will involve characterization of wild type enzyme and mutants by genetic and biochemical means, isolation of putative mutants with altered sequence specificity, and isolation and characterization of an isoschizomer of TagI. Wild type endonuclease has been overproduced and purified as will a "canonical site nicking" mutant and an "allosteric activation" mutant. These will be characterized for sequence specific binding, dissociation, canonical and "star" site cleavage using a variety of DNA oligonucleotide and plasmid substrates. In collaboration with Dr. John Anderson, purified TagI will be co-crystallized with its cognate oligonucleotide for determination of its three dimensional structure. Mutants will be generated using two-codon insertion mutagenesis, oligonucleotide directed mutagenesis, and saturation mutagenesis. Two in vivo screens will be developed and may help identify mutants with altered sequence specificity. These mutants should help define amino acid residues involved in sequence-specific hydrogen bonds to the major groove, other sequence-specific contacts, allosteric activation of enzyme activity, and protein subunit interactions. The properties and sequence of an isoschizomer that recognizes DNA with similar structural motifs as TagI will be determined. The amino acid sequence of the isoschizomer might point to regions of functional homology in TagI necessary for sequence specific DNA recognition.

本项目的长期目标是了解 蛋白质对DNA的序列特异性识别。 DNA识别 控制许多代谢活动，包括基因激活， 阻遏、位点特异性重组和DNA起始 复制的 了解这些蛋白质如何工作将提供 新蛋白质特异性的合理设计框架 可能具有医学和药理学应用。 研究的特殊蛋白质是嗜热限制酶 核酸内切酶TagI。 提出了一种TagI识别模型 它的同源序列。 进一步完善这一模式将 包括通过遗传学方法表征野生型酶和突变体 和生物化学手段，分离假定的突变体， 序列特异性，以及分离和表征一种 TagI的异构体。 野生型核酸内切酶已被过量生产和纯化， “典型位点切口”突变体和“变构激活” 变种人 这些将被表征为序列特异性结合， 解离、正则和“星星”位点裂解， DNA寡核苷酸和质粒底物。 合作 与John安德森博士一起，纯化的TagI将与 其同源寡核苷酸用于测定其三种 空间结构 使用双密码子插入诱变产生突变体， 寡核苷酸定向诱变和饱和诱变。 将开发两种体内筛选，可能有助于识别突变体 改变了序列特异性 这些变种人应该能帮忙 定义参与序列特异性氢的氨基酸残基 与大沟的键合，其它序列特异性接触， 酶活性和蛋白质亚基的变构活化 交互. 识别DNA的异构体的性质和序列 具有与TagI相似的结构基序的蛋白质。 的 异构体的氨基酸序列可能指向 序列特异性DNA所必需的TagI的功能同源性 识别.