title: Cointegrate-resolution of toluene-catabolic transposon Tn4651: determination of crossover site and the segment required for full resolution activity.
abstract: 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.
authors.affiliation: Department of Environmental Life Sciences, Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Sendai 980-8577, Japan. hyano@uidaho.edu
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Yano
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Genka
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Ohtsubo
authors.forename: Yuji
authors.initials: Y
authors.lastname: Nagata
authors.forename: Eva M
authors.initials: EM
authors.lastname: Top
authors.forename: Masataka
authors.initials: M
authors.lastname: Tsuda
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D003434:Crossing Over, Genetic; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D024202:Electrophoretic Mobility Shift Assay; D005822:Genetic Vectors / Q000235:genetics; D055029:Inverted Repeat Sequences; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016958:Pseudomonas putida / Q000235:genetics*; D011995:Recombination, Genetic; D014050:Toluene / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D014050:Toluene