title: Trans catalysis in Tn5 transposition.
abstract: Synaptic complexes in prokaryotic transposons occur when transposase monomers bind to each of two specific end-binding sequences and then associate to bring the proteins and the two ends of the transposon together. It is within this complex of proteins and DNA that identical catalytic reactions are carried out by transposase on each of the ends of the transposon. In this study, we perform in vitro transposition reactions by combining the methylated inside end (IE(ME)) biased hyperactive Tn5 transposase, Tnp sC7 version 2.0, and the outside end (OE) biased hyperactive Tn5 transposase, Tnp EK/LP, with plasmid DNA containing a transposon defined by one IE(ME) and one OE. These two proteins cooperate to facilitate double end cleavage of the transposon from the plasmid and conversion into transposition products via strand transfer. When one of the hyperactive Tnps is replaced with a catalytically inactive version containing the mutation EA326 (DDE mutant), the predominant reaction product is a linearized plasmid resulting from single end cleavage. Restriction analysis of these linear products reveals that cleavage is occurring on the end distal to that which is bound by the transposase with an intact active site or in trans. Similar in vitro experiments performed with precut transposons and a supercoiled target plasmid demonstrated that the strand transfer reaction is also facilitated by a trans active DDE motif.
authors.affiliation: Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
authors.forename: T A
authors.initials: TA
authors.lastname: Naumann
authors.forename: W S
authors.initials: WS
authors.lastname: Reznikoff
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D019895:Transposases / Q000235:genetics / Q000378:metabolism*
chemical_list: D019895:Transposases