title: Unlocking Tn3-family transposase activity in vitro unveils an asymetric pathway for transposome assembly.
abstract: The Tn3 family is a widespread group of replicative transposons that are notorious for their contribution to the dissemination of antibiotic resistance and the emergence of multiresistant pathogens worldwide. The TnpA transposase of these elements catalyzes DNA breakage and rejoining reactions required for transposition. It also is responsible for target immunity, a phenomenon that prevents multiple insertions of the transposon into the same genomic region. However, the molecular mechanisms whereby TnpA acts in both processes remain unknown. Here, we have developed sensitive biochemical assays for the TnpA transposase of the Tn3-family transposon Tn4430 and used these assays to characterize previously isolated TnpA mutants that are selectively affected in immunity. Compared with wild-type TnpA, these mutants exhibit deregulated activities. They spontaneously assemble a unique asymmetric synaptic complex in which one TnpA molecule simultaneously binds two transposon ends. In this complex, TnpA is in an activated state competent for DNA cleavage and strand transfer. Wild-type TnpA can form this complex only on precleaved ends mimicking the initial step of transposition. The data suggest that transposition is controlled at an early stage of transpososome assembly, before DNA cleavage, and that mutations affecting immunity have unlocked TnpA by stabilizing the protein in a monomeric activated synaptic configuration. We propose an asymmetric pathway for coupling active transpososome assembly with proper target recruitment and discuss this model with respect to possible immunity mechanisms.
authors.affiliation: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Emilien
authors.initials: E
authors.lastname: Nicolas
authors.affiliation: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Cédric A
authors.initials: CA
authors.lastname: Oger
authors.affiliation: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Nathan
authors.identifier: 0000-0001-6969-3894
authors.initials: N
authors.lastname: Nguyen
authors.affiliation: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Michaël
authors.initials: M
authors.lastname: Lambin
authors.affiliation: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Amandine
authors.initials: A
authors.lastname: Draime
authors.affiliation: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium.
authors.forename: Sébastien C
authors.initials: SC
authors.lastname: Leterme
authors.affiliation: Laboratoire de Microbiologie et Génétique Moléculaires, CNRS UMR5100, F-31062 Toulouse, France.
authors.forename: Michael
authors.identifier: 0000-0002-0292-6662
authors.initials: M
authors.lastname: Chandler
authors.affiliation: Institut des Sciences de la Vie, Université Catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium; bernard.hallet@uclouvain.be.
authors.forename: Bernard F J
authors.initials: BF
authors.lastname: Hallet
mesh_terms: D004247:DNA / Q000737:chemistry; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation; D019895:Transposases / Q000737:chemistry* / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D004247:DNA; C473502:Tn3 transposase; D019895:Transposases
keywords: DDE/D transposase; DNA transposition; Tn3; target immunity; transpososome