Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition. We report the three-dimensional structure of prokaryotic Tn5 transposase complexed with Tn5 transposon end DNA determined to 2.3 angstrom resolution. The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites. This structure provides a molecular framework for understanding many aspects of transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA.

This communication reports the development of an efficient in vitro transposition system for Tn5. A key component of this system was the use of hyperactive mutant transposase. The inactivity of wild type transposase is likely to be related to the low frequency of in vivo transposition. The in vitro experiments demonstrate the following: the only required macromolecules for most of the steps in Tn5 transposition are the transposase, the specific 19-bp Tn5 end sequences, and target DNA; transposase may not be able to self-dissociate from product DNAs; Tn5 transposes by a conservative "cut and paste" mechanism; and Tn5 release from the donor backbone involves precise cleavage of both 3 and 5 strands at the ends of the specific end sequences.Transposition is a process that gives rise to a variety of genomic rearrangements such as insertions, deletions, inversions, and chromosome fusions (1). Because transposition can play a profound role in genome evolution and in a variety of genetic diseases, how this process occurs and how its frequency is determined is of considerable interest. Bacterial transposons, such as Tn5, are convenient tools for studying these two questions.The transposition process can be understood in the context of the following model (2, 3). The first step in transposition involves the sequence-specific binding of the transposon-encoded transposase (Tnp) 1 to the specific end sequences that define the ends of the transposable element. The second step involves formation of a synaptic complex in which the ends of the transposable element are brought together through Tnp oligomerization. In some well studied cases such as Tn10 (4, 5), synaptic complex formation is facilitated by one or more host proteins. The third step involves a Tnp-mediated nucleolytic attack on the phosphodiester bonds adjacent to the ends of the transposable element. The precise nature of these cleavages (whether just the 3Ј ends of the transposon are liberated or both the 3Ј and the 5Ј ends and the exact location of the cleavage in the case of 5Ј end release) is a special property of the system being studied. The fourth step involves Tnp binding to the target DNA sequence (target capture). Different transposons demonstrate various degrees of sequence specificity at this step. The fifth step involves a concerted nucleophilic attack by the transposon 3Ј-OH ends on phosphodiester bonds in both strands of

DNA transposition is an important biological phenomenon that mediates genome rearrangements, inheritance of antibiotic resistance determinants, and integration of retroviral DNA. Transposition has also become a powerful tool in genetic analysis, with applications in creating insertional knockout mutations, generating gene-operon fusions to reporter functions, providing physical or genetic landmarks for the cloning of adjacent DNAs, and locating primer binding sites for DNA sequence analysis. DNA transposition studies to date usually have involved strictly in vivo approaches, in which the transposon of choice and the gene encoding the transposase responsible for catalyzing the transposition have to be introduced into the cell to be studied (microbial systems and applications are reviewed in ref. 1). However, all in vivo systems have a number of technical limitations. For instance, the transposase must be expressed in the target host, the transposon must be introduced into the host on a suicide vector, and the transposase usually is expressed in subsequent generations, resulting in potential genetic instability. A number of in vitro transposition systems (for Tn5, Tn7, Mu, Himar1, and Ty1) have been described, which bypass many limitations of in vivo systems. For this purpose, we have developed a technique for transposition that involves the formation in vitro of released Tn5 transposition complexes (TransposomesTM) followed by introduction of the complexes into the target cell of choice by electroporation. In this report, we show that this simple, robust technology can generate high-efficiency transposition in all tested bacterial species (Escherichia coli, Salmonella typhimurium, and Proteus vulgaris) We also isolated transposition events in the yeast Saccharomyces cerevisiae.

The initial chemical steps in Tn5 transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3 hydrolytic nick by transposase. The free 3OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3 and 5 strands without massive protein/DNA rearrangements.Genomic DNA is known to undergo a variety of rearrangements, including inversions, deletions, duplications, and translocations. Transposition is one of the ways by which these rearrangements occur. The transposon Tn5 is a 5.8-kilobase pair prokaryotic, composite transposon consisting of two inverted repeats, IS50L and IS50R, that flank genes encoding antibiotic resistance. IS50R codes for the Tn5 transposase (Tnp), the protein catalyzing all the steps in transposition. Each IS50 repeat is bracketed by two different 19-bp 1 end sequences, termed the outside end (OE) and the inside end (IE), that are specifically recognized by Tn5 transposase ( Fig. 1) (reviewed in Refs. 1 and 2). Transposition of the full Tn5 element requires two OEs, whereas transposition of an IS50 element requires one OE and one IE. In vitro, Tn5 transposition requires only a hyperactive mutant transposase, such as EK54/LP372 Tnp, Mg 2ϩ , transposon DNA defined by two inverted 19-bp end sequences, and target DNA (3). Transposition frequency can be increased by using the mosaic end sequence (ME), a hyperactive, synthetic end sequence that is a hybrid of the OE and IE (Fig. 1) (4).Tn5 is a member of the "cut and paste" family of transposons that includes Tn10 and Tn7. Although the transposition reactions vary in details, they follow the same basic mechanism. Transposase binds to the transposon DNA at the end recognition sequences. Then, the end sequences are brought together via transposase oligomerization to form a complex nucleoprotein structure, 2 termed a synaptic complex (5, 6). Once a stable synaptic complex has been formed, the transposon end sequence-donor DNA boundary can be cleaved to release a pair of 3ЈOHs at the ends, which are then used in the next chemical step, strand transfer ( Fig. 2A). Strand transfer occurs via a one step transesterification (7,8) in which the 3ЈOHs attack phosphodiester bonds in the target DNA in a staggered fashion. For Tn5, the staggered attack leads to 9-bp gaps flanking the integrated transposon. The gaps are presumed to be filled in and ligated in the cell, and this leads to 9-bp duplications of the target sequence flanking the transposon (9, ...

This communication reports an analysis of Tn5͞IS50 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 Tn5͞IS50 bound transposase protomers, was deduced by analyzing all insertion sites. The consensus Tn5͞IS50 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 Tn5͞IS50 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.

RNA-seq has been widely adopted as a gene-expression measurement tool due to the detail, resolution, and sensitivity of transcript characterization that the technique provides. Here we present two transposon-based methods that efficiently construct high-quality RNA-seq libraries. We first describe a method that creates RNA-seq libraries for Illumina sequencing from double-stranded cDNA with only two enzymatic reactions. We generated high-quality RNA-seq libraries from as little as 10 pg of mRNA (~1 ng of total RNA) with this approach. We also present a strand-specific RNA-seq library construction protocol that combines transposon-based library construction with uracil DNA glycosylase and endonuclease VIII to specifically degrade the second strand constructed during cDNA synthesis. The directional RNA-seq libraries maintain the same quality as the nondirectional libraries, while showing a high degree of strand specificity, such that 99.5% of reads map to the expected genomic strand. Each transposon-based library construction method performed well when compared with standard RNA-seq library construction methods with regard to complexity of the libraries, correlation between biological replicates, and the percentage of reads that align to the genome as well as exons. Our results show that high-quality RNA-seq libraries can be constructed efficiently and in an automatable fashion using transposition technology.

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A synaptic complex of Tn5 transposase with an extended outside end DNA duplex was prepared and crystallized, and its crystal structure was determined in an effort to reveal the role of metal ions in catalysis. Two Mn2+ ions bound to the active site when a single nucleotide of donor DNA was added to the 3' end of the transferred strand. Marked conformational changes were observed in the DNA bases closest to the active site. The position of the metal ions and the conformational changes of the DNA provide insight into the mechanism of hairpin formation and cleavage, and is consistent with a two-metal model for catalysis.