Catalysts of Strand Exchange by Tn3 Resolvase

McIlwraith, Michael John (1995) Catalysts of Strand Exchange by Tn3 Resolvase. PhD thesis, University of Glasgow.

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A novel methodology has been developed to test the mechanism of strand exchange by Tn3 resolvase. Photocrosslinkable substrate molecules were synthesised by incorporating photoactivatable nucleotide analogues into res site I oligonucleotides and then ligating these double-stranded oligonucleotides into supercoiled plasmid molecules in vitro. It was then possible to covalently attach a resolvase subunit to a specific half-site of the modified site I by irradiating bound samples with UV laser light, and to assay if the crosslinked complex was catalytically active. The nucleotide analogues 4-thiothymidine and 6-thiodeoxyguanosine photocrosslinked to resolvase, but were unsuitable because they apparently induced structural anomalies in supercoiled molecules. The use of 5-iodouridine in conjunction with a 325 nm HeCd laser allowed efficient photochemical crosslinking of resolvase to a synthetic site I, and the photoreactivity was retained when the site was incorporated into supercoiled substrate molecules. Using this method, it was shown that fixing a subunit of resolvase to either the left or the right end of site I does not affect its ability to participate in the catalysis of recombination. These results demonstrate for the first time that the resolvase C-terminal domain-DNA interaction is maintained throughout the recombination reaction, supporting a 'subunit rotation' or 'DNA-mediated' mechanism of strand exchange. A separate series of experiments is also described in which an attempt was made to distinguish between the 'subunit rotation' and 'DNA-mediated' models of strand exchange. The distinction between the models was based on the assumption that there is a requirement for a ligated recombinant intermediate during a 'double round' knotting reaction in a 'DNA-mediated' mechanism but not in a 'subunit rotation' mechanism. Using substrates with mutations at the centre of site I, substrates with closely spaced res sites, and substrates containing mismatched base pairs at the centre of site I, it was demonstrated that a fully ligated, mismatched recombinant is not a necessary intermediate in the resolvase-mediated knotting reaction. To investigate the role of the 'AT' central dinucleotide of site I in DNA cleavage and strand exchange, resolution substrates were constructed with base pairs added or deleted at the centre of site I. It was found that in substrates with an additional base pair at the centre of site I, the resolvase-induced cleavages are not fixed at 2 bp apart (although this is preferred) but can be 3 bp apart. Furthermore, cleavage is not specifically at the AT sequence; rather, the top strand cleavage position seems to be fixed relative to the right end of site I, perhaps due to an interaction between the subunits at the right end of site I and those bound at sites II and III of res.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Marshall Stark
Keywords: Biochemistry
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-75620
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 19:16
Last Modified: 19 Nov 2019 19:16

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