Engineering Site-Specific DNA Endonucleases with Novel Properties

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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.