Transposon-encoded site-specific recombination

Symington, Lorraine S (1982) Transposon-encoded site-specific recombination. PhD thesis, University of Glasgow.

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The closely related transposable elements, Tn3 and , share significant DNA sequence homology and functional organization. These elements have been shown to encode interchangeable resolvase proteins to mediate resolution of obligatory transpositional cointegrates. There is no apparent complementation between these elements for the tnpA gene function. To analyze the mechanism of resolvase-mediated recombination, the Tn3-encoded tnpR gene was cloned into a high expression plasmid vector. This allowed large amounts of resolvase to be synthesized, thus aiding purification of the protein. The purified protein was subsequently used for biochemical analysis of the resolution reaction and for DNase I footprinting experiments. A number of small plasmid substrates were constructed containing two res sites in direct or inverted orientation. In vitro resolution reactions were assayed by gel electrophoresis to detect the formation of interlocked circles (catenates) which appeared to be the major reaction product. The resolution reaction requires only resolvase and a supercoiled substrate containing two directly repeated sites, under the appropriate ionic conditions; the reaction is independent of host factors or an external energy supply. Catenates are always formed during resolution suggesting that this may be a direct consequence of the reaction mechanism" Substrates containing varying lengths of DNA separating res sites have differing efficiencies of resolution in vitro, the reaction proceeds with greatest efficiency when the length of DNA between (vii) res sites is least . This data, in conjunction with the observed preference for two directly repeated ves sites in cis, has led to the proposal of a "tracking" model for resolvase-mediated recombination. Resolvase has been shown to bind specifically and nonspecifically to DNA. Specific binding, resulting from tight association of resolvase with the target site, was investigated by sequence protection from DNase I. This revealed three binding sites for resolvase within the res region. The sequences of the protected sites conform to the consensus sequence proposed for other regulatory DNA binding proteins.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: David Sherratt
Keywords: Biochemistry
Date of Award: 1982
Depositing User: Enlighten Team
Unique ID: glathesis:1982-72483
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 11 Jun 2019 11:06
Last Modified: 11 Jun 2019 11:06

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