Structural and Biophysical Characterization of Engineered Homing Endonucleases (C

工程化归巢核酸内切酶 (C) 的结构和生物物理表征

• 批准号: 7858482
• 负责人: BARRY L. STODDARD
• 金额: $ 43.2万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-26 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7858482
• 关键词: Bacteriophages; Base Pairing; Behavior; Binding; Biochemical; Biological Assay; Biological Models; Biological Process; Calorimetry; Cells; Complex; Computing Methodologies; DNA; DNA Binding; DNA-Binding Proteins; Data; Deoxyribonuclease I; Discrimination; Engineering; Entropy; Enzymes; Eubacterium; Event; Family; Gene Targeting; Genes; Genome; Genome engineering; Goals; Heating; Homing; Immunoglobulin Somatic Hypermutation; In Vitro; Individual; Laboratories; Measures; Methods; Mutation; Paper; Pattern; Production; Property; Proteins; Protocols documentation; Publishing; Reagent; Resolution; Roentgen Rays; Series; Site; Specificity; Structure; Thermodynamics; Titrations; Validation; Variant; Vertebral column; base; cost; endonuclease; gene therapy; improved; interest; novel; research study; scaffold; three dimensional structure

Homing endonucleases are extraordinarily specific DNA-binding proteins, acting specifically at individual sites within a host genome. These proteins are under instense study for the purpose of engineering single chain gene-specific reagents to be used for gene therapy and other applications. Over the past 10 years, we have determined the structure and mechanisms of representatives form all known families of homing endonucleases, found respectively in phage, eubacteria, archae, and single cell eukarya. In addition, we have described the creation of homing endonuclease variants that act at noncognate sites. These constructs have been generated using both bacterial selection strategies and compuational methods, both of which target enzyme residues that directly contact DMA basepairs. In either case, such experiments have produced endonucleases that display shifted DMA recognition properties, but at the cost of reduced site-discrimination abilities. We hypothesize that in order to completely reprogram the DNA recognition specificity of a homing endonuclease, without a reduction in site discrimination, the resculpting of protein-DNA contacts must be combined with the selection of structural mutations in the nearby enzyme scaffold that "fine-tune" the protein -DNA interaface of each novel cognate complex. The goal of overall Specific Aim 1 of the Northwest Genome Engineering Consortium is to accomplish this task by combining somatic hypermutation of the endonuclease scaffold, computational redesign and selection of DNA contacts, and biochemical/biophysical characterization of the resulting endonuclease constructs. In our component of the consortium's activities, we will be responsible for the following aims: 1. We will determine the in vitro site specificity profile of the novel endonuclease construcst using two related methods to directly visualize cleavage of DNA target variants and to quantitate specificity at each base pair. 2. We will determine the thermodynamic signature of cognate and non-cognate site recognition for redesigned homing endonucleases, using isothermal titration calorimetry (ITC). 3. We will determine the three-dimensional structure of novel endonuclease-DNA cognate pairs at high resolution, and will characterize (a) the effect of enzyme scaffold mutations on backbone structure, and 9b) the accuracy of computational redesign predictions within the protein-DNA interface.

归巢核酸酶是特定特异性的DNA结合蛋白，专门起作用于个体 宿主基因组中的站点。这些蛋白质正在概念研究中，目的是工程单。 用于基因治疗和其他应用的链基因特异性试剂。在过去的10年中，我们 已经确定了代表的结构和机制，形成了所有已知家庭的家庭 核酸内切酶，分别在噬菌体，Eubacteria，Archae和单细胞Eukarya中发现。另外，我们 已经描述了在非认知位点起作用的归核核酸酶变体的创建。这些结构 已经使用细菌选择策略和构造方法生成 靶向直接接触DMA底部的酶残基。无论哪种情况，此类实验都产生了 核酸内切酶显示出改变DMA识别属性，但以降低现场歧视为代价 能力。我们假设为了完全重新编程归居的DNA识别特异性 核酸内切酶，如果不减少现场歧视，蛋白质-DNA接触的撤土必须为 结合在附近的酶支架中选择“微调”蛋白质的结构突变 - 每个新型同源复合物的DNA界面。西北的总体特定目标1的目标 基因组工程联盟是通过结合体细胞超夸张来完成这项任务 核酸内切酶支架，计算重新设计和DNA接触的选择以及生化/生物物理 结果的核酸内切酶构建体的表征。 在财团活动的组成部分中，我们将负责以下目的： 1。我们将使用两个确定新核酸内切酶约束的体外位点特异性曲线 直接可视化DNA靶标变体的切割并定量特异性的相关方法 基对。 2。我们将确定同源和非认知位点识别的热力学特征 使用等温滴定量热法（ITC），重新设计的归巢核酸酶。 3。我们将确定高核酸内切酶-DNA同源对的三维结构 分辨率，并将表征（a）酶支架突变对主链结构的影响，而9b） 蛋白质-DNA界面内计算重新设计预测的准确性。