Year: 2015
Type: dissertation
Abstract: The zinc finger recombinase (ZFR) is a chimeric enzyme system for use in targeted genomic editing.The ZFR is comprised of a recombinase catalytic domain, which is able to catalyse recombination reactions between DNA molecules, and a zinc finger array DNAbinding domain, which is able to target the enzyme to a desired genetic sequence.Currently the ZFR is in an early stage of development and will require several crucial improvements before it can be adopted as a useful genome editing tool by researchers.Two major challenges involve two important parameters of ZFR-catalysed integration reactions: specificity of the orientation of the integration, and stability of the integration.Currently, the ZFR system is unable to select the orientation of integrations, and because the recombination reactions are reversible, the integrations are not stable (and, in fact, are stochastically disfavoured).This project aimed to impart the ZFR system with the ability to perform stable, orientation-specific integrations.In order to achieve the project aims, the experiments of this project sought to generate new pairs of ZFR mutants that were differentially modified at either their catalytic domain or DNA-binding domain, and characterize their behaviour in an E. colibased recombination assay.The overall strategy was to exploit the interactions within the protein-protein interfaces of the ZFR tetramer to produce selective compatibility, and to generate differences in enzyme activity when two mutants (one active, and one inactive or less active) were either paired as heterodimers or as homodimers.During ZFR recombination reactions heterodimers rearrange to form homodimers, and thus the production of a significant difference in activity between heterodimers and homodimers represents a recombination reaction directionality bias.Both the catalytic domain modification and DNA-binding domain modification approaches proved able to produce the desired bias in the directionality of ZFR recombination reactions, which is predicted to lead to both stability of an integration, and specificity of integration orientation through a stochastic process.Of particular note, was a strategy utilizing a heterodimer that consisted of one ZFR subunit targeted specifically to the DNA, paired with a recombinase subunit (with its native DNA-binding domain) targeted non-specifically to the DNA.The difference in activity between these subunits paired in heterodimer and homodimer configurations appeared to produce a completely irreversible recombination reaction
Author: Jeffrey Louis Kentner
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2439110476