Structural and Biophysical Characterization of Engineered Homing Endonucleases (C

归巢内切酶是一种非常特异的DNA结合蛋白，它专门作用于个体 宿主基因组中的位置。这些蛋白质正在进行内部研究，目的是为了制造单一的工程。 用于基因治疗和其他应用的链状基因特异性试剂。在过去的10年里，我们 已经确定了来自所有已知归宿家族的代表的结构和机制 核酸内切酶，分别存在于噬菌体、真核细菌、古细菌和单细胞真核细胞中。此外，我们 已经描述了作用于非同源位点的归巢核酸内切酶变体的创建。这些构造 都是使用细菌选择策略和计算方法产生的，这两种方法都是 直接接触DMA基对的靶标酶残留物。在这两种情况下，这样的实验都产生了 显示转移的DMA识别属性的内切酶，但代价是减少了位点歧视 超能力。我们假设，为了完全重新编程寻的DNA识别特异性 内切酶，在不减少位点歧视的情况下，蛋白质-DNA接触的重新计算必须是 结合对附近酶支架结构突变的选择，这些突变可以对蛋白质进行微调 -每个新的同源复合体的DNA界面。西北地区总体目标1的目标 基因组工程联盟是通过将体细胞的超突变结合在一起来完成这一任务 核酸内切酶支架，DNA接触的计算重新设计和选择，以及生化/生物物理 由此产生的核酸内切酶构建物的特征。 在财团活动的我们组成部分中，我们将负责实现以下目标： 1.我们将用两种方法测定新型内切酶构建体的体外位点特异性 直接可视化DNA靶变异体切割并定量检测每个变异体的特异性的相关方法 碱基对。 2.我们将确定同源和非同源位置识别的热力学特征 重新设计了归巢核酸内切酶，使用等温滴定热法(ITC)。 3.确定新型核酸内切酶-DNA同源对的三维结构 分辨率，并将表征(A)酶支架突变对骨架结构的影响，以及9b) 蛋白质-DNA界面内计算重新设计预测的准确性。

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.