Site-specific recombination at res sites containing DNA-binding sequences for both Tn21 and Tn3 resolvases.
Tn3 and gamma delta resolvases catalyse site-specific recombination at res sites from Tn3 but not at Tn21 res sites. Tn21 resolvase has no activity at Tn3 sites and acts only at Tn21 sites. In both Tn3 and Tn21, res had three binding sites for the cognate resolvases; the cross-over site, I; and the accessory sites II and III, from which the bound proteins may stabilize the synaptic complex by protein-protein interactions. In this study hybrid res sites were made by replacing either II or III in the Tn21 res site with the equivalent sequence from Tn3. Plasmids containing either a hybrid and a wild-type Tn21 res site, or two hybrid sites, were tested for recombination. Relative to the reaction with two wild-type sites, recombination by Tn21 resolvase was reduced by replacing II at one res site and it was reduced further by replacing II at both loci but, in both cases, Tn21 recombination was enhanced by Tn3 or gamma delta resolvases. Very few of the amino acid on the external surface of gamma delta resolvases are conserved in Tn21. Moreover, mutants of gamma delta resolvase with defective protein-protein interactions also enhanced Tn21 recombination at this hybrid site. The resolvase at II thus seems not to be involved in protein-protein interactions and its main role may be to bend the DNA to the required structure. The replacement of III in the Tn21 site with Tn3 sequence also reduced recombination by Tn21 resolvase, especially when both loci carried the alteration but, in contrast to before, Tn3 or gamma delta resolvases now inhibited the Tn21 reaction. Recombination thus seems to require identical proteins at I and III, perhaps to allow for protein-protein interactions.
Department of Biochemistry, University of Bristol, U.K.
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Soultanas
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Oram
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Halford
D001483:Base Sequence; D001665:Binding Sites; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D011485:Protein Binding; D011995:Recombination, Genetic; D013379:Substrate Specificity; D019895:Transposases; D045564:Transposon Resolvases
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D009713:Nucleotidyltransferases; C473490:Tn21 resolvase; D019895:Transposases; D045564:Transposon Resolvases