Dissection of the Tn3 Resolution Site

[摘要] Resolvase recognises three different sites within res: all three are necessary for recombination in vivo and in vitro, under standard reaction conditions. However, when resolvase In vitro reaction conditions were altered, recombination between a wt-res site and an isolated crossover site (i. e. lacking subsites II and III) proceeded at a reduced efficiency. In these substrates, resolvase disregarded the relative orientation of the crossover site, but still selected the resolution event. The resolution products were simply catenated. Resolvase thus recognises the crossover site as functionally symmetrical. Replacing the crossover site within wt-res with a perfectly symmetrical subsite I (sym-res) resulted in the normal left-to-right alignment of crossover sites for recombination, even for intermolecular recombination between two linear sym-res substrates. Therefore, resolvase uses subsites II and III to determine the polarity of res. When subsites II and III were removed from both res partners, no recombination products were detected. To investigate the effect of FIS on resol vase-mediated recombination, the enhancer site, sis. was cloned into resolvase substrates. Although FIS and sis are required to stimulate inversion by the related Gin, Hin and Cin invertases, they did not appear to have any effect on the recombination properties of isolated res crossover sites; resolvase reactions with other combinations of wt-res and deleted res sites were also unaffected by these accessory proteins and sites. Substrates were made to test whether resolvase acting at subsites II and III can direct a Gin-mediated resolution event between gix sites (i.e. ges' site recombination). No recombination between ges sites was observed in vivo when resolvase and Gin were provided in trans. In collaboration with C. Koch, (Berlin) in vitro recombination between ges sites was tested using a FIS-independent mutant Gin protein capable of recombining directly repeated gix sites; when resolvase was present recombination between ges sites by the mutant Gin was prevented. This may be interpreted as a consequence of synapsis of subsites II and III by resolvase inhibiting Gin-mediated recombination. Subsites II and III alone were also shown to delay recombination between certain pairs of wt-res sites in a multi-res site substrate. This result also suggests that subsites II and III are sufficient for synapsis. Individual res subsites, and combinations of res subsites, on DNA fragments displayed distinctive retarded complexes in resolvase gel binding assays. By using this assay and a set of circularly permuted DNA fragments, resol vase-induced bending of subsite I was demonstrated. Two complexes per subsite were stabilised in the gel, suggesting that resolvase can occupy a subsite in two steps. No severely retarded complexes were trapped by the gel assay that would be indicative of a higher protein-DNA structure, i.e. a synaptic intermediate. Therefore, intermolecular synapsis of sites by resolvase appears to be difficult to capture in the gel assay.

[发布机构] University:University of Glasgow

[关键词] Genetics