Mutants of Tn3 Resolvase

Arnold, Patricia Hazel (1997) Mutants of Tn3 Resolvase. PhD thesis, University of Glasgow.

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Prior to this work, no mutants of Tn3 resolvase had been isolated. To construct a variety of mutants, a suitable plasmid for mutagenesis purposes was created. This plasmid, pAT5, contains unique restriction sites along two thirds of the length of the resolvase open reading frame. This allows double stranded oligonucleotides containing mutations to be cloned into the resolvase gene. pAT5 was also designed to be compatible with the galK screen used to investigate the in vivo properties of the mutants. The in vivo assay was based on change of colony colour on MacConkey agar plates when the cells were unable to utilise galactose, due to the deletion of a plasmid-encoded galK gene by resolvase-catalysed recombination. Two specific mutants of Tn5 resolvase were made, Y6F and E124Q. Random mutagenesis of the E-helix of resolvase created a library of mutants, which was screened in vivo for ability to resolve test plasmids containing either two res sites, or two isolated copies of binding site I of res (the crossover site) or one site I and one res. The mutant resolvase D102Y was isolated, which promoted the reaction between res v site I in vivo. In order to purify these and any subsequent mutants, a new purification procedure was developed. This is a denaturing protocol which has been shown to be reproducible for wild-type resolvase and the mutants mentioned above. A new expression plasmid, using a promoter recognised by the phage T7 RNA polymerase was created, resulting in higher yields of resolvase per gram of cells. In vitro analysis of Y6F resolvase showed that the protein was recombinationally inactive on a wild type substrate, but retained binding ability as well as cleavage and religation activities. This supports the theory that resolvase linkage to the DNA during recombination is by a phosphoseryl, and not a phosphotyrosyl linkage as in topoisomerases. L69F mutant resolvase, inadvertently created during the construction of the mutant selection system, retains recombination activity, but displays topoisomerase activity and aberrations in binding. The Tn3 resolvase mutant E124Q was observed to display enhanced cleavage activity, but unlike the gammadelta E124Q resolvase mutant does not show the ability to resolve substrates containing partial res sites. The mutant isolated using the in vivo screen, D102Y resolvase, was shown to display the same phenotype in vitro as in vivo, and catalysed recombination between res and site I. This protein also catalyses intermolecular reactions, and may be described as having a "hyperactive" phenotype. The isolation of a mutant resolvase with this phenotype is a major step towards the isolation of a mutant able to catalyse a recombination reaction between two site I's.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Marshall Stark
Keywords: Genetics, Molecular biology
Date of Award: 1997
Depositing User: Enlighten Team
Unique ID: glathesis:1997-74844
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 15:54
Last Modified: 27 Sep 2019 15:54

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