Electronic Thesis and Dissertation Repository


Master of Science




Prof. David R. Edgell


Engineering nucleases is important to the advancement of genetic engineering and gene therapy approaches. Engineering requires a knowledge of which residues are contributing to each function of the nuclease. The residues which contribute to cleavage specificity of the I-TevI nuclease domain (ND) are unknown. I suspect that some of these contributions derive from the ND, thus my null hypothesis is that mutation of the ND will not alter the substrates this enzyme can cut. I have mutagenised the I-TevI nuclease domain and using directed evolution I have isolated mutations which were characterised in vivo and in vitro. These mutations permit cleavage of otherwise cleavage resistant substrates, indicating that the ND does contribute to cleavage specificity. Mutations which provided the greatest increase in activity against cleavage resistant substrates (K26R, T95S, and Q158R) were combined into a single relaxed specificity nuclease domain which exhibits 1.2-5-fold improved cleavage of resistant substrates.

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