Engineering Site-Specific DNA Endonucleases with Novel Properties

归巢核酸内切酶是由移动的DNA元件编码的酶，并且在所有生命王国中发现。 它们通过催化宿主基因组内的双链断裂来启动这些元件在种群内和物种之间的转移。 该项目的目标是设计具有新功能的归巢核酸内切酶，以利用这些蛋白质的极端靶特异性。 PI-SceI归巢核酸内切酶识别1300万碱基对酵母基因组内的单个靶位点。本研究的一个目的是修饰PI-SceI以产生识别不同靶位点的改变的特异性核酸内切酶。 这将通过基于PI-SceI/DNA复合物的X射线晶体结构的合理设计与组合遗传选择策略相结合来完成。 另一个目的是阐明如何改变特异性发生在自然界中通过分析PI-SceI类似物从其他酵母菌株。 这将揭示归巢核酸内切酶的特异性决定簇如何随时间演变。 该项目还将涉及将笼状配体引入PI-SceI中，以产生新型人工酶，其核酸内切活性可以在紫外线照射下触发。 这些变体将允许酶活性的时间和空间调节。更广泛的影响：所述项目的一个重要好处是，它们将成为培训研究生和博士后科研人员的基础。 PI-SceI和靶向特定基因组序列的新型位点特异性核酸内切酶的可用性将促进复杂生物体中DNA修复的研究，并提供基因靶向方法所需的分子工具。 此外，即将开发的光活化归巢核酸内切酶可能成为重要的商业技术。

Homing endonucleases are enzymes that are encoded by mobile DNA elements, and are found in all kingdoms of life. They initiate the transfer of these elements within a population and between species by catalyzing double-strand breaks within the host genome. The goal of this project is to engineer homing endonucleases with novel functions that harness the extreme target specificity of these proteins. The PI-SceI homing endonuclease recognizes a single target site within the 13 million base-pair yeast genome. One objective of this research is to modify PI-SceI to create altered specificity endonucleases that recognize different target sites. This will be done by coupling rational-design, based on the X-ray crystal structure of the PI-SceI/DNA complex, with a combinatorial genetic selection strategy. Another aim is to elucidate how altered specificity occurs in nature by analyzing PI-SceI analogues from other yeast strains. This will reveal how the specificity determinants of homing endonucleases evolve over time. The project will also involve introducing caged ligands into PI-SceI in order to generate novel artificial enzymes whose endonucleolytic activities can be triggered in response to ultraviolet irradiation. These variants will permit the temporal and spatial regulation of the enzymatic activity. Broader impact: An important benefit of the described projects will be that they will form the basis for the training of graduate students and post-doctoral fellows in scientific research. The availability of PI-SceI and of novel site-specific endonucleases that target specific genomic sequences will facilitate the study of DNA repair in complex organisms and provide the molecular tools required for gene targeting methods. Moreover, the photoactivated homing endonuclease that will be developed may become an important commercial technology.