Dyson, Paul J.
(1984)
The mechanism of site-specific recombination encoded by TN3.
PhD thesis, University of Glasgow.
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Abstract
The bacterial ampicillin resistance transposon Tn3 encodes a site-specific recombination system which mediates the second step in its transposition pathway: the resolution of cointegrates (Arthur and Sherratt (1979) Mol.gen.Genet. 175, 267-274). It vitro analysis has shown that this recombination system is simple: dependent on the transposon encoded resolvase protein acting at res, a site required in cis(Reed (1981) Cell 25, 713-719). Resolvase binds at res at three sites (Grindley et al (1982) Cell 30, 713-719). res was defined to a region of 139bp by in vivo analysis, which confirms that the extent of DNA bound by resolvase in vitro constitutes res. Truncated res regions have been analysed for resolvase binding and function. The results confirm the importance of a sequence determinant for resolvase binding, suggest non-cooperative binding at each of the three sites, but cooperativity for binding at both half-sites of the individual binding sites. In terms of function a truncated res region was shown to have secondary site, activity in that it could recombine with res-wt but not efficiently with itself. The difference in primary and secondary res sites is the presence of resolvase binding site III in the former. This binding site can be reintroduced at a different position to a truncated res to partially reconstitute primary site activity. The normal cointegrate substrate contains directly repeated copies of res. Inversion substrates, containing two inverted res regions, were analysed for recombination. Inversion is approximately 50 times less efficient than resolution. This rate is comparable to that of resolution of a substrate containing directly repeated secondary res-regions. These results, taken in context with evidence from other workers on both Tn3 encoded site-specific recombination and bacteriophage lambda site-specific recombination, suggest that the mechanism by which two res regions are brought together prior to recombination is determined by the higher-order structure formed by resolvase and res.
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