ABSTRACT Tn5 was one of the first transposons to be identified ( 10 ). As a result of Tn5's early discovery and its simple macromolecular requirements for transposition, the Tn5 system has been a very productive tool for studying the molecular mechanism of DNA transposition. These studies are of broad value because they offer insights into DNA transposition in general, because DNA transposition is a useful model with which to understand other types of protein-DNA interactions such as retroviral DNA integration and the DNA cleavage events involved in immunoglobulin gene formation, and because Tn5-derived tools are useful adjuncts in genetic experimentation.
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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.Proceedings of the National Academy of Sciences 09/2000; 97(16):8944-9. · 9.74 Impact Factor
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ABSTRACT: The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans. In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay. Two mutations in the carboxyl terminus were isolated repeatedly. The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans. The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans. Combining both mutations increases Tnp activity 80-fold. Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh. Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers. Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization. In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein.Genes & Development 11/1994; 8(19):2363-74. · 12.44 Impact Factor
Article: Tn5/IS50 target recognition.[show abstract] [hide abstract]
ABSTRACT: This communication reports an analysis of Tn5/IS50 target site selection by using an extensive collection of Tn5 and IS50 insertions in two relatively small regions of DNA (less than 1 kb each). For both regions data were collected resulting from in vitro and in vivo transposition events. Since the data sets are consistent and transposase was the only protein present in vitro, this demonstrates that target selection is a property of only transposase. There appear to be two factors governing target selection. A target consensus sequence, which presumably reflects the target selection of individual pairs of Tn5/IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5/IS50 target site is A-GNTYWRANC-T. However, we observed that independent insertion sites tend to form groups of closely located insertions (clusters), and insertions very often were spaced in a 5-bp periodic fashion. This suggests that Tn5/IS50 target selection is facilitated by more than two transposase protomers binding to the DNA, and, thus, for a site to be a good target, the overlapping neighboring DNA should be a good target, too. Synthetic target sequences were designed and used to test and confirm this model.Proceedings of the National Academy of Sciences 10/1998; 95(18):10716-21. · 9.74 Impact Factor