Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

David Edgell

Abstract

The ability to manipulate complex genomes in a precise manner is essential for studying biological processes in model systems, engineering plant strains for agriculture, or advancing human cellular therapies to treat diseases. Genomic alterations are most efficient when a double-strand DNA break is introduced at the loci where the modification is desired. Different classes of naturally occurring DNA endonucleases, including homing endonucleases, have therefore been explored as candidates for genome modification studies as they target long stretches of DNA. Homing endonucleases are mobile genetic elements whose biological role is to introduce site-specific double-strand breaks into naïve genomes, ultimately resulting in the selfish propagation of their own genes. Consequently, homing endonucleases are an ideal enzymatic system whose natural properties can be exploited to manipulate genes.

In the present studies, I examine the cleavage mechanism of GIY-YIG family homing endonucleases, as until now the method by which they hydrolyze DNA has remained poorly understood. Using the GIY-YIG homing endonuclease I-BmoI as a model system, I investigate the amino acid, nucleotide, and divalent metal ion requirements of the GIY-YIG nuclease domain to generate a double-strand break. I specifically test models of hydrolysis by which enzymes with a single active site could nick both strands of DNA, and determine that I-BmoI functions as a monomer throughout the reaction pathway. Furthermore, I demonstrate that the nuclease domain itself has weak binding affinity, is tethered to DNA by a high affinity binding domain, and must reposition across each strand through a series of protein and substrate conformational changes to facilitate DNA hydrolysis.

To explore the relevance of GIY-YIG homing endonucleases as genome editing reagents, I fused the nuclease domain of I-TevI to three different re-targetable DNA-binding platforms utilized in the field. The engineered nucleases developed within the present studies are mechanistically distinct from established technologies, as they function as monomers and cleave DNA at a preferred sequence motif. I therefore envision that the engineered GIY-YIG nucleases may circumvent complications associated with established technologies, and provide an alternative and potentially safer set of genome editing reagents.


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