Structural and Biophysical Characterization of Engineered Homing Endonucleases (C

归巢核酸内切酶是非常特异性的 DNA 结合蛋白，专门作用于个体 宿主基因组内的位点。为了工程化单一的目的，这些蛋白质正在被深入研究 用于基因治疗和其他应用的链基因特异性试剂。过去10年，我们 确定了所有已知归巢家族代表的结构和机制 核酸内切酶，分别存在于噬菌体、真细菌、古细菌和单细胞真核生物中。此外，我们 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 已经使用细菌选择策略和计算方法生成，这两种方法 直接接触 DMA 碱基对的目标酶残基。无论哪种情况，此类实验都产生了 显示转移的 DMA 识别特性的核酸内切酶，但以减少位点歧视为代价 能力。我们假设，为了完全重新编程归巢的 DNA 识别特异性 核酸内切酶，在不减少位点区分的情况下，蛋白质-DNA接触的重塑必须是 结合附近酶支架中结构突变的选择，“微调”蛋白质 -每个新型同源复合物的DNA界面。西北地区总体具体目标1的目标 基因组工程联盟将通过结合体细胞超突变来完成这项任务 核酸内切酶支架、DNA 触点的计算重新设计和选择以及生物化学/生物物理 所得核酸内切酶构建体的表征。 在我们联盟活动的组成部分中，我们将负责实现以下目标： 1. 我们将使用两种方法确定新型核酸内切酶构建体的体外位点特异性概况 相关方法可直接可视化 DNA 靶标变体的切割并定量每个变体的特异性 碱基对。 2. 我们将确定同源和非同源位点识别的热力学特征 使用等温滴定量热法（ITC）重新设计了归巢核酸内切酶。 3. 我们将在高温下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) 酶支架突变对主链结构的影响，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.