Active site sharing and subterminal hairpin recognition in a new class of DNA transposases

Many bacteria harbor simple transposable elements termed insertion sequences (IS). In Helicobacter pylori, the chimeric IS605 family elements are particularly interesting due to their proximity to genes encoding gastric epithelial invasion factors. Protein sequences of IS605 transposases do not bear the hallmarks of other well-characterized transposases. We have solved the crystal structure of full-length transposase (TnpA) of a representative member, ISHp608. Structurally, TnpA does not resemble any characterized transposase; rather, it is related to rolling circle replication (RCR) proteins. Consistent with RCR, Mg2+ and a conserved tyrosine, Tyr127, are essential for DNA nicking and the formation of a covalent intermediate between TnpA and DNA. TnpA is dimeric, contains two shared active sites, and binds two DNA stem loops representing the conserved inverted repeats near each end of ISHp608. The cocrystal structure with stem-loop DNA illustrates how this family of transposases specifically recognizes and pairs ends, necessary steps during transposition.     

In vitro reconstitution of a single-stranded transposition mechanism of IS608

Bacterial insertion sequences (IS) play an important role in restructuring their host genomes. IS608, from Helicobacter pylori, belongs to a newly recognized and widespread IS group with a unique transposition mechanism. We have reconstituted the entire set of transposition cleavage and strand transfer reactions in vitro and find that, unlike any other known transposition system, they strictly require single-strand DNA. TnpA, the shortest identified transposase, uses a nucleophilic tyrosine for these reactions. It recognizes and cleaves only the IS608 "top strand." The results support a transposition model involving excision of a single-strand circle with abutted left (LE) and right (RE) IS ends. Insertion occurs site specifically 3' to conserved and essential TTAC tetranucleotide and appears to be driven by LE. This single-strand transposition mode has important implications not only for dispersion of IS608 but also for the other members of this very large IS family.     

Transposition of ISHp608, member of an unusual family of bacterial insertion sequences

ISHp608 from Helicobacter pylori is active in Escherichia coli and represents a recently recognised group of insertion sequences. Its transposase and organisation suggest that it transposes using a different mechanism to that of other known transposons. The IS was shown to excise as a circular form, which is accompanied by the formation of a resealed donor plasmid backbone. We also demonstrate that TnpA, which is less than half the length of other transposases, is responsible for this and for ISHp608 transposition. Transposition was shown to be site specific: both insertion and transposon excision require a conserved target, 5'TTAC. Deletion analysis suggested that potential secondary structures at the left and right ends are important for transposition. In vitro TnpA bound both ends, showed a strong preference for a specific single-stranded DNA and introduced a single-strand break on the same strand at each end. Although many of the characteristics of ISHp608 appear similar to rolling-circle transposons, there are differences suggesting that, overall, transposition occurs by a different mechanism. The results have permitted the formulation of several related models.     

Mechanism of IS200/IS605 family DNA transposases: activation and transposon-directed target site selection

The smallest known DNA transposases are those from the IS200/IS605 family. Here we show how the interplay of protein and DNA activates TnpA, the Helicobacter pylori IS608 transposase, for catalysis. First, transposon end binding causes a conformational change that aligns catalytically important protein residues within the active site. Subsequent precise cleavage at the left and right ends, the steps that liberate the transposon from its donor site, does not involve a site-specific DNA-binding domain. Rather, cleavage site recognition occurs by complementary base pairing with a TnpA-bound subterminal transposon DNA segment. Thus, the enzyme active site is constructed from elements of both protein and DNA, reminiscent of the interdependence of protein and RNA in the ribosome. Our structural results explain why the transposon ends are asymmetric and how the transposon selects a target site for integration, and they allow us to propose a molecular model for the entire transposition reaction.     

A unique right end-enhancer complex precedes synapsis of Mu ends: the enhancer is sequestered within the transpososome throughout transposition

Assembly of the Mu transpososome is dependent on interactions of transposase subunits with the left (L) and right (R) ends of Mu and an enhancer (E). We have followed the order and dynamics of association of these sites within a series of transpososomes prior to and during formation of a three-site complex (LER), engagement of Mu ends by the transposase active site (type 0 complex), cleavage of the ends (type I complex) and their transfer to target DNA (type II complex). LER appears to be preceded by a two-site complex (ER) where E and R are interwrapped twice, as in the mature transpososome. At each stage thereafter, the overall topology of five DNA supercoils is retained: two between E and R, one between E and L and two between L and R. However, L-R interactions within LER appear to be flexible. Unexpectedly, the enhancer was seen to persist within the transpososome through cleavage and strand transfer of Mu ends to target DNA.     

Interactions of phage Mu enhancer and termini that specify the assembly of a topologically unique interwrapped transpososome

The higher-order DNA-protein complex that carries out the chemical steps of phage Mu transposition is organized by bridging interactions among three DNA sites, the left (L) and right (R) ends of Mu, and an enhancer element (E), mediated by the transposase protein MuA. A subset of the six subunits of MuA associated with their cognate sub-sites at L and R communicate with the enhancer to trigger the stepwise assembly of the functional transpososome. The DNA follows a well-defined path within the transpososome, trapping five supercoil nodes comprising two E-R crossings, one E-L crossing and two L-R crossings. The enhancer is a critical DNA element in specifying the unique interwrapped topology of the three-site LER synapse. In this study, we used multiple strategies to characterize Mu end-enhancer interactions to extend, modify and refine those inferred from earlier analyses. Directed placement of transposase subunits at their cognate sub-sites at L and R, analysis of the protein composition of transpososomes thus obtained, and their characterization using topological methods define the following interactions. R1-E interaction is essential to promote transpososome assembly, R3-E interaction contributes to the native topology of the transpososome, and L1-E and R2-E interactions are not required for assembly. The data on L2-E and L3-E interactions are not unequivocal. If they do occur, either one is sufficient to support the assembly process. Our results are consistent with two R-E and perhaps one L-E, being responsible for the three DNA crossings between the enhancer and the left and right ends of Mu. A 3D representation of the interwrapped complex (IW) obtained by modeling is consistent with these results. The model reveals straightforward geometric and topological relationships between the IW complex and a more relaxed enhancer-independent V-form of the transpososome assembled under altered reaction conditions.     

Single-stranded DNA intermediates in IS91 rolling-circle transposition

IS91 displays a number of characteristics unique among insertion sequence (IS) elements, suggesting that it transposes by a novel mechanism called rolling-circle (RC) transposition. We reported previously that IS91 transposase (TnpA) amino acid sequence shares a series of five conserved signatures with A proteins of RC replicating phages, including a pair of invariant tyrosines that catalyse two successive transesterification reactions during replication initiation and termination. To analyse their role in IS91 transposition, we constructed a series of TnpA derivatives in which the invariant Tyr-249 and/or Tyr-253 were mutated to either phenylalanine or serine. Mutation of either tyrosine resulted in complete loss of transposition activity in vivo. This result was taken as a first new line of evidence that TnpA is a functional analogue of phiX174 phage A protein. Secondly, RC replication plasmids and phages accumulate single-stranded DNA (ssDNA) intermediates as a result of uncoupled leading and lagging DNA strand synthesis. Using a plasmid carrying an IS91-derived IRLkan-IRR transposable cassette, in which the left (IRL)- and right (IRR)-terminal sequences of IS91 flank a kanamycin resistance gene (kan), we demonstrated the in vivo formation of two new DNA species after induction of transposase expression. The first was a circular ssDNA that contained the transposable cassette covalently joined at its exact termini, whereas the second was a double-stranded circle of the same element. When this experiment was repeated using the mutant transposases described above, the ssDNA and dsDNA intermediates could not be observed, indicating that the integrity of both Y249 and Y253 was essential for their appearance. The presence of ssDNA intermediate products is the first biochemical evidence for a RC mechanism of IS91 transposition.     

Structuring the bacterial genome: Y1-transposases associated with REP-BIME sequences

REPs are highly repeated intergenic palindromic sequences often clustered into structures called BIMEs including two individual REPs separated by short linker of variable length. They play a variety of key roles in the cell. REPs also resemble the sub-terminal hairpins of the atypical IS200/605 family of insertion sequences which encode Y1 transposases (TnpA(IS200/IS605)). These belong to the HUH endonuclease family, carry a single catalytic tyrosine (Y) and promote single strand transposition. Recently, a new clade of Y1 transposases (TnpA(REP)) was found associated with REP/BIME in structures called REPtrons. It has been suggested that TnpA(REP) is responsible for REP/BIME proliferation over genomes. We analysed and compared REP distribution and REPtron structure in numerous available E. coli and Shigella strains. Phylogenetic analysis clearly indicated that tnpA(REP) was acquired early in the species radiation and was lost later in some strains. To understand REP/BIME behaviour within the host genome, we also studied E. coli K12 TnpA(REP) activity in vitro and demonstrated that it catalyses cleavage and recombination of BIMEs. While TnpA(REP) shared the same general organization and similar catalytic characteristics with TnpA(IS200/IS605) transposases, it exhibited distinct properties potentially important in the creation of BIME variability and in their amplification. TnpA(REP) may therefore be one of the first examples of transposase domestication in prokaryotes.     

Tn10 transposase mutants with altered transpososome unfolding properties are defective in hairpin formation

Transposition reactions take place in the context of higher-order protein-DNA complexes called transpososomes. In the Tn10 transpososome, IHF binding to an "outside end" creates a bend in the DNA that allows the transposase protein to contact the end at two different sites, the terminal and subterminal binding sites. Presumably this helps to stabilize the transposase-end interaction. However, the DNA loop that is formed must be unfolded at a later stage in order for the transposon to integrate into other DNA molecules. It has been proposed that transpososome unfolding also plays a role in transposon excision. To investigate this possibility further, we have isolated and characterized transposase mutants with altered transpososome unfolding properties. Two such mutants were identified, R182A and R184A. Both mutants fail to carry out hairpin formation, an intermediate step in transposon excision, specifically with outside end-containing substrates. These results support the idea that transpososome unfolding and excision are linked. Also, based on the importance of residues R182 and R184 in transpososome unfolding, we propose a new model for the Tn10 transpososome, wherein both DNA ends of the transpososome make subterminal contacts with transposase.     

The organization of the outside end of transposon Tn5.

The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.     

The positive and negative regulation of Tn10 transposition by IHF is mediated by structurally asymmetric transposon arms

The Tn10 transpososome has symmetrical components on either side: there are two transposon ends each of which has binding sites for a monomer of transposase and an IHF heterodimer. The DNA bending activity of IHF stimulates assembly of an intermediate with tightly folded transposon ends in which transposase has additional ‘subterminal’ DNA contacts, located distal to the IHF site. These subterminal contacts are required to activate later steps in the reaction. Quantitative hydroxyl radical footprinting and gel retardation unfolding experiments show that the transpososome is fundamentally asymmetric, despite having identical components on either side. Major differences between the transposon ends define α and β sides of the complex. IHF can dissociate from the transposon arm on the β side of the complex in the absence of metal ion. However, IHF is locked onto the α side of the complex, probably by the subterminal transposase contacts, until released by a metal ion-dependent conformational change. Later in the reaction, IHF inhibits target interactions. Using a very short transposon arm, target interactions are demonstrated at a saturating IHF concentration. This suggests that inhibition of target interactions is due to steric hindrance of the target binding site by a single IHF-folded transposon arm.     

Conformational isomerization in phage Mu transpososome assembly: effects of the transpositional enhancer and of MuB.

Initiation of phage Mu DNA transposition requires assembly of higher order protein-DNA complexes called Mu transpososomes containing the two Mu DNA ends and MuA transposase tetramer. Mu transpososome assembly is highly regulated and involves multiple DNA sites for transposase binding, including a transpositional enhancer called the internal activation sequence (IAS). In addition, a number of protein cofactors participate, including the target DNA activator MuB ATPase. We investigated the impact of the assembly cofactors on the kinetics of transpososome assembly with the aim of deciphering the reaction steps that are influenced by the cofactors. The transpositional enhancer IAS appears to have little impact on the initial pairing of the two Mu end segments bound by MuA. Instead, it accelerates the post-synaptic conformational step(s) that converts the reversible complex to the stable transpososome. The transpososome assembly stimulation by MuB does not require its stable DNA binding activity, which appears critical for directing transposition to sites distant from the donor transposon.     

Sequences essential for IS50 transposition. The first base-pair

Sequences near the ends of the insertion element IS50 are essential for its transposition, probably because they serve as sites upon which the IS50-encoded transposase protein acts. To determine if these essential sequences include the first base-pair at each end of IS50 we generated 5'C to 5'G transversions at these positions. Each mutation reduced the transposition frequency to 1% to 2% of wild-type. DNA sequence analyses showed that the mutant 5'G is preserved during transposition.     

Progressive structural transitions within Mu transpositional complexes

Assembly of the Mu transpososome is dependent on specific binding sites for the MuA transposase near the ends of the phage genome. MuA also contacts terminal nucleotides but only upon transpososome assembly, and base-specific recognition of the terminal nucleotides is critical for assembly. We show that Mu ends lacking the terminal 5 bp can form transpososomes, while longer DNA substrates with mutated terminal nucleotides cannot. The impact of the mutations can be suppressed by base mismatches near the end of Mu. Deletion of the flanking strands or mutation of the terminal nucleotides has differential effects on the cleavage and strand transfer reactions. These results show that the terminal nucleotides control the assembly and activation of transpososomes by influencing conformational changes around the active site.     

Isolation and characterization of IS1409, an insertion element of 4-chlorobenzoate-degrading Arthrobacter sp. strain TM1, and development of a system for transposon mutagenesis

A new insertion element of 1,449 bp with 25-bp perfect terminal repeats, designated IS1409, was identified in the chromosome of 4-chlorobenzoate-degrading Arthrobacter sp. strain TM1 NCIB12013. Upon insertion, IS1409 causes a target duplication of 8 bp. IS1409 carries only a single open reading frame of 435 codons encoding the transposase TnpA. Both TnpA and the overall organization of IS1409 are highly similar to those of some related insertion elements of the ISL3 group (J. Mahillon and M. Chandler, Microbiol. Mol. Biol. Rev. 62:725--774, 1998). IS1409 was also found in other 4-chlorobenzoate-degrading Arthrobacter strains and Micrococcus luteus. Based on IS1409, a series of transposons carrying resistance genes for chloramphenicol and gentamicin were constructed. These transposons were used to demonstrate transposition events in vivo and to mutagenize Arthrobacter sp. strains.     

Target DNA bending is an important specificity determinant in target site selection in Tn10 transposition

The bacterial transposon Tn10 inserts preferentially into specific DNA sequences. DNA footprinting and interference studies have revealed that the Tn10-encoded transposase protein contacts a large stretch of target DNA ( approximately 24 bp) and that the target DNA structure is deformed upon incorporation into the transpososome. Target DNA deformation might contribute significantly to target site selection and thus it is of interest to further define the nature of this deformation. Circular permutation analysis was used to demonstrate that the target DNA is bent upon its incorporation into the transpososome. Two lines of evidence are presented that target DNA bending is an important event in target site selection. First, we demonstrate a correlation between increased target site usage and an increased level of target DNA bending. Second, transposase mutants with relaxed target specificity are shown to cause increased target DNA bending relative to wild-type transposase. This latter observation provides new insight into how relaxed specificity may be achieved. We also show that Ca(2+) facilitates target capture by stabilizing transposase interactions with sequences immediately flanking the insertion site. Ca(2+) could, in theory, exert this effect by stabilizing bends in the target DNA.     

Linearization and transposition of circular molecules of insertion sequence IS3.

IS3 transposase has been shown to promote production of characteristic circular and linear IS3 molecules from the IS3-carrying plasmid; IS3 circles have the entire IS3 sequence with terminal inverted repeats, IRL and IRR, which are separated by a three base-pair sequence originally flanking either end in the parental plasmid, whereas linear IS3 molecules have three nucleotide overhangs at their 5' ends. Here, we showed that a plasmid carrying an IS3 derivative, which is flanked by different sequences at both ends, generated IS3 circles and linear IS3 molecules owing to the action of transposase. Cloning and sequencing analyses of the linear molecules showed that each had the same 5'-protruding three nucleotide overhanging sequences at both ends, suggesting that the linear molecules were not generated from the parental plasmid by the two double-strand breaks at both end regions of IS3. The plasmid carrying IS3 with a two base-pair mutation in the terminal dinucleotide, which would be required for transposase to cleave the 3' end of IS3, could still generate linear molecules as well as circles. Plasmids bearing an IS3 circle were cleaved by transposase and gave linear molecules with the same 5'-protruding three nucleotide overhanging sequences. These show that the linear molecules are generated from IS3 circles via a double-strand break at the three base-pair intervening sequence. Plasmids carrying an IS3 circle with the two base-pair end mutation still were cleaved by transposase, though with reduced efficiencies, suggesting that IS3 transposase has the ability to cleave not only the 3' end of IS3, but a site three nucleotides from the 5' end of IS3. IS3 circles also were shown to transpose to the target plasmids. The end mutation almost completely inhibited this transposition, showing that the terminal dinucleotides are important for the transfer of the 3' end of IS3 to the target as well as for the end cleavage.     

Control of IS911 target selection: how OrfA may ensure IS dispersion

IS911 transposition involves a closed circular insertion sequence intermediate (IS-circle) and two IS-encoded proteins: the transposase OrfAB and OrfA which regulates IS911 insertion. OrfAB alone promotes insertion preferentially next to DNA sequences resembling IS911 ends while the addition of OrfA strongly stimulates insertion principally into DNA targets devoid of the IS911 end sequences. OrfAB shares its N-terminal region with OrfA. This includes a helix-turn-helix (HTH) motif and the first three of four heptads of a leucine zipper (LZ). OrfAB binds specifically to IS911 ends via its HTH whereas OrfA does not. We show here: that OrfA binds DNA non-specifically and that this requires the HTH; that OrfA LZ is required for its multimerization; and that both motifs are essential for OrfA activity. We propose that these OrfA properties are required to assemble a nucleoprotein complex committed to random IS911 insertion. This control of IS911 insertion activity by OrfA in this way would assure its dispersion.     

Determinants for hairpin formation in Tn10 transposition

Tn10 transposition involves the formation of a hairpin intermediate at the transposon termini. Here we show that hairpin formation exhibits more stringent DNA sequence requirements at the terminal two base pairs than either transpososome assembly or first strand nicking. We also observe a significant DNA distortion at the terminal base pairs upon transpososome assembly by chemical nuclease footprinting. Interestingly, mutations at these positions do not necessarily inhibit the formation of the distortion. However, it remains a possibility that the inhibitory effect of these mutations is due to a defect in protein-DNA interactions subsequent to this deformation. Terminal base pair mutations also inhibited strand transfer, providing evidence that transposase interactions with the terminal residues on both 'transferred' and 'non-transferred' strands are important for hairpin formation. We also demonstrate that mutation of a highly conserved tyrosine residue that is a component of the YREK motif, Y285, results in a phenotype comparable to that of the terminal base pair mutations. In contrast, a mutation at another conserved position, W265, is shown to relax the specificity of the hairpin formation reaction.     

Two Classes of Tn10 Transposase Mutants That Suppress Mutations in the Tn10 Terminal Inverted Repeat

Tn10 transposition requires IS10 transposase and essential sequences at the two ends of the element. Mutations in terminal basepairs 6-13 confer particularly strong transposition defects. We describe here the identification of transposase mutations that suppress the transposition defects of such terminus mutations. These mutations are named ``SEM'''' for suppression of ends mutations. All of the SEM mutations suppress more than a single terminus mutation and thus are not simple alterations of transposase/end recognition specificity. The mutations identified fall into two classes on the basis of genetic tests, location within the protein and nature of the amino acid substitution. Class I mutations, which are somewhat allele specific, appear to define a small structural and functional domain of transposase in which hydrophobic interactions are important at an intermediate stage of the transposition reaction, after an effective interaction between the ends but before transposon excision. Class II mutations, which are more general in their effects, occur at a single residue in a small noncritical amino-terminal proteolytic domain of transposase and exert their affects by altering a charge interaction; these mutations may affect act early in the reaction, before or during establishment of an effective interaction between the ends.