Engineering Site-Specific DNA Endonucleases with Novel Properties

归巢内切酶是由可移动的DNA元件编码的酶，存在于所有生命王国中。它们通过催化宿主基因组中的双链断裂，启动这些元素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标特异性。Pi-SCEI定位内切酶识别1300万个碱基对的酵母基因组中的单个靶点。这项研究的一个目标是修改PI-SCEI以创建识别不同靶点的改变的特异性内切酶。这将通过将基于PI-SCEI/DNA复合体的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实现。另一个目的是通过分析其他酵母菌株的PI-SCEI类似物来阐明自然界中发生改变的特异性是如何发生的。这将揭示归巢内切酶的特异性决定因素如何随着时间的推移而演变。该项目还将在PI-SCEI中引入笼形配体，以产生可在紫外线照射下触发内切核活性的新型人工酶。这些变异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，它们将成为培养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内切酶的可获得性将促进复杂生物中DNA修复的研究，并为基因打靶方法提供所需的分子工具。此外，即将开发的光活化归巢内切酶可能成为一项重要的商业化技术。

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.