Synapsis of res Sites by Tn3 Resolvase

Watson, Mark Albert (1994) Synapsis of res Sites by Tn3 Resolvase. PhD thesis, University of Glasgow.

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Protein-DNA complexes formed by synapsis of res sites by Tn5 resolvase were isolated by agarose gel electrophoresis. Prior treatment with a protein crosslinking reagent was required in order to isolate synaptic complexes with a characteristic gel mobility following restriction endonuclease cleavage within one or both domains of a negatively supercoiled plasmid DNA substrate containing direct repeats of the Tn5 res site. An intramolecular synaptic complex was observed, following treatment with various protein crosslinking reagents, in a yield that represented between 20 and 50% of the input substrate DNA when Mg2+ was omitted from the reaction. The gel mobility of this species was shown to vary in a predictable manner when the site of restriction endonuclease cleavage was altered. There was no evidence of an equivalent synaptic complex when a negatively supercoiled plasmid containing a single Tn5 res site and a single Tn2i res site in direct repeat orientation was assayed in the presence of both resolvase proteins. Despite the apparent stability of the crosslinked Tn5 resolvase synaptic complex during agarose gel electrophoresis, attempts to recover it intact following elution from the gel were unsuccessful. The gel mobility assay also indicated that a synaptic interaction, stabilised by protein crosslinking, occurred between two res sites present in inverted repeat orientation on a negatively supercoiled plasmid DNA molecule. Similarly, intramolecular synapsis between two copies of the isolated accessory subsites (subsites II and III) of res was demonstrated. Although intramolecular synapsis was observed between an isolated copy of subsite I and an intact res site, there was no evidence of synapsis between two copies of the isolated subsite I of res oriented as direct repeats on a negatively supercoiled plasmid. Intramolecular synapsis of two copies of the intact res site present in direct repeat orientation on either nicked or linearised plasmid substrate was observed. In addition, intermolecular synapsis was seen; interactions between plasmids containing two res sites (involving two, three, and four res sites on two DNA molecules) were characterised. The two-plasmid four-site intermolecular S5maptic complex was seen in by far the highest yield when two intact res sites were in direct repeat orientation on a supercoiled DNA molecule. Surprisingly, the two-res-site intermolecular sjraaptic complex, clearly in evidence when the plasmid substrate assayed contained two res sites (whether supercoiled, nicked, or linearised) was barely detectable when interacting supercoiled DNA molecules contained only a single res site. In the presence of Mg2+, a protein-DNA complex representing s5niapsis of the res sites in the (-2) catengine product of the resolution reaction was isolated following treatment with a protein crosslinking reagent and restriction endonuclease cleavage. It is not known whether this species represented an intermediate in the resolution reaction, trapped prior to dissociation of the synapse, or whether it was due to re-synapsis of catenane product. Although re-synapsis of catenane was demonstrated, it appeared too inefficient to account for the yield of the product synaptic complex typically seen in the 'forward reaction.' Assays of the rate of s3niapsis indicated rapid formation of the intramolecular synaptic complex. The ensuing gradual decline in the amount of this substrate synaptic complex was concomitant with an increase in the amount of the product synaptic complex. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Marshall Stark
Keywords: Genetics
Date of Award: 1994
Depositing User: Enlighten Team
Unique ID: glathesis:1994-74640
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 27 Sep 2019 17:24
Last Modified: 27 Sep 2019 17:24

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