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，特异性作用于个体 宿主基因组中的一个位点。这些蛋白质正在进行深入研究，目的是工程化单克隆抗体。 链基因特异性试剂用于基因治疗和其他应用。在过去的10年里，我们 已经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细胞中。另外我们 已经描述了作用于非同源位点的归巢核酸内切酶变体的产生。这些构建体 已经使用细菌选择策略和计算方法生成，两者都 靶向直接接触DMA碱基对的酶残基。无论哪种情况，这样的实验都产生了 显示移位的DNA识别特性的核酸内切酶，但以降低的位点区分为代价 能力.我们假设，为了完全重新编程归巢的DNA识别特异性， 在不减少位点区分的情况下，蛋白质-DNA接触的重新雕刻必须是 结合在附近的酶支架中选择结构突变来“微调”蛋白质， - 每个新同源复合物的DNA界面。西北地区总体具体目标1 基因组工程联合会将通过结合体细胞超突变来完成这一任务。 核酸内切酶支架，计算重新设计和DNA接触的选择，以及生物化学/生物物理 对所得内切核酸酶构建体进行表征。 在联合体的活动中，我们将负责实现以下目标： 1.我们将使用两种酶来确定新型核酸内切酶构建体的体外位点特异性概况。 直接可视化DNA靶变体的切割并定量每个位点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点识别的热力学特征， 重新设计的归巢核酸内切酶，使用等温滴定量热法（ITC）。 3.我们将在高温下确定新型内切酶-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，和9 b） 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.