Tn3-family transposon Tn4651 from Pseudomonas putida mt-2 plasmid pWW0 carries two divergently transcribed genes, tnpS and tnpT, for cointegrate-resolution. While tnpS encodes a tyrosine recombinase, tnpT encodes a protein that shows no homology to any other characterized protein. The Tn4651 resolution site was previously mapped within the 203-bp fragment that covered the tnpS and tnpT promoter region. To better understand the molecular mechanisms underlying the Tn4651 cointegrate-resolution, we determined the extent of the functional resolution site (designated the rst site) of Tn4651 and the location of the crossover site for the cointegrate-resolution. Deletion analysis of the rst region localized the fully functional rst site to a 136-bp segment. The analysis of the site-specific recombination between Tn4651 rst and a rst variant from the Tn4651-related transposon, Tn4661, indicated that the crossover occurs in the 33-bp inverted repeat region, which separates the 136-bp functional rst site into the tnpS- and tnpT-proximal segments. Electrophoretic mobility shift assays demonstrated specific binding of TnpT to the 20-bp inverted repeat region in the tnpT-proximal segment. The requirement for accessory sequences on both sides of the crossover site and the involvement of the unique DNA-binding protein TnpT suggest that the Tn4651-specified resolution system uses a different mechanism than other known resolution systems. Furthermore, comparative sequence analysis for Tn4651-related transposons revealed the occurrence of DNA exchange at the rst site among different transposons, suggesting an additional role of the TnpS-TnpT-rst system in the evolution of Tn4651-related transposons.
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