CHEMICAL BIOLOGY

DNA Transposition: Topics in Chemical Biology

 

Philippe Rousseau and Michael Chandler, Laboratoire de Microbiologie et Genetique Moleculaires, CNRS and University Paul Sabatier, Toulouse, France

doi: 10.1002/9780470048672.wecb639

 

DNA transposable elements are a ubiquitous and highly diverse group of mobile genetic elements capable of moving within and between genomes. Despite their diversity, only a limited number of chemical mechanisms, which are catalyzed by enzymes called transposases, are used to promote this movement. DNA transposases can be classified according to these chemistries. We outline present knowledge that concerns the mechanisms adopted by the five different types of transposase identified to date: the DDE-, Y-, S-, Y2-, and Y1-transposases. The DDE and Y1 enzymes are perhaps the best characterized, whereas the data available for the Y- and S-transposases suggest that they use similar mechanisms to their closely related cousins, the Y- and the S- site-specific recombinases.

 

Transposable elements (TE) are segments of DNA capable of moving from one locus in a genome to another, or from one genome to another, using mechanisms that do not depend on large regions of sequence homology between the TE and the target DNA site. Their effects were observed first in plants in the middle of the twentieth century (1), and they were characterized at the molecular and mechanistic levels, largely in bacteria, in the last three decades (2). With the birth of whole genome sequencing, a growing recognition has occurred of the ubiquity of TEs, of their diversity, and of their importance in shaping both eukaryote and prokaryote genomes and in influencing genome function. In some cases, their numbers are so high that it is tempting to consider that, together with bacteriophages, plasmids, and mobile introns, they form part of a genomic “ecosystem.” In the prokaryotes alone, nearly 2000 different insertion sequences (ISs; the simplest form of autonomous TEs) have been identified (IS-finder: www-is.biotoul.fr). TEs have been classified in various ways (see http://bioinformatics.org/wikiposon/doku.php). For example, in eukaryotes, a major division can be drawn between those elements that transpose via an RNA intermediate (Class I; retroviruses, retrotransposons) and those that transpose via a DNA intermediate (Class II; DNA transposons). Nevertheless, perhaps the most pertinent classification, for the purposes of this article, that deals exclusively with DNA transposons is based on the reaction mechanisms that they have adopted for their movement (3). These reactions involve cleavage of the DNA at the ends of the TE in the donor DNA molecule and transfer of these ends into a target DNA. They are catalyzed by a TE-encoded enzyme, the transposase. In the special case of retroviruses, the (DNA) provirus is liberated from its donor site not by DNA cleavages but by transcription to generate an RNA copy. During the viral lifecycle, this provirus is subsequently reverse-transcribed into a double-strand DNA genomic copy. This copy is then processed by an enzyme that resembles a transposase, the retroviral integrase or IN, which also assures subsequent integration into the host genome. Currently, the following five types of DNA transposase have been recognized: the DDE enzymes (most are identified so far) (3, 4); the tyrosine transposases (related to tyrosine site-specific recombinases of the phage λ Int family) (2); the S-transposases (related to serine site-specific recombinases of the γ δ resolvase family) (2); the Y2-transposases (related to Rep proteins involved in rolling circle replication and to Relaxases, which are involved in conjugative gene transfer in bacteria) (5); and, finally, the Y1-transposases (also related to Rep and Relaxase proteins). The Y1-transposases were discovered only a few years ago (6-8), and it seems likely that other types of enzyme will be identified in the future. Each class of enzymes catalyzes a distinct chemistry that we describe below.

DDE Transposases and Integrases

The DDE enzymes are named for their characteristic triad of acidic amino acids (aspartate and glutamate). These enzymes bind in a sequence-specific way to the ends of the TE that generally carry terminal inverted repeat sequences and include the transposase recognition sequence. The simplest TEs, such as bacterial ISs, tend to carry only single transposase binding sites at each end, whereas other more complex transposable elements may carry arrays of such binding sites. These arrays may be arranged in different patterns at each end and provide a means for distinguishing one end from the other. This arrangement could be involved, for example, in regulating transposon gene expression or in forming an asymmetric synaptic complex. It could lead to end-specific ordered cleavages or strand transfers that would determine the order of events that lead to integration.

Many DDE transposases carry a DNA sequence-specific binding domain in their N-terminal regions and at least one domain involved in multimerization. Generally, the catalytic domain is located in the C-terminal part of the protein. The DDE domain is by far the most studied and best understood catalytic motif involved in transposition. It is found in many different types of transposon from retroviruses to Tc-Mariner, bacterial ISs, and transposons (4).

Analysis of transposition of TE with DDE transposases has shown that it is regulated in vivo (9). Where analyzed, a high level of coordination between transposase expression and its activity has been observed. Presumably, this coordination prevents excessive damage to the host genome by high levels of transposon-induced rearrangements. For many bacterial ISs, a weak transposase promoter is located within one end that is also recognized and bound by the transposase itself. Such interactions provide an autoregulatory mechanism for transposase expression.

Because, in prokaryotes, transcription and translation occur in the same cell compartment, the N-terminal DNA recognition domain has been suggested to impose an additional level of regulation, so-called activity in cis. It is thought that this domain could fold and bind the IS end correctly before the entire protein has been translated. Generally, full-length transposases bind poorly to IS ends. In several cases, it has been observed that the C-terminal transposase end inhibits transposase binding. This would favor transposase binding to the IS from which it has been produced.

Several additional layers of regulation occur at the level of transposase expression. One of these, specific to some mobile genetic elements, is the transient assembly of a strong transposase promoter during transposition. This is relatively common in bacterial insertion sequences. Many IS transpose using a circular transposition intermediate and, in addition to a generally weak endogenous transposase promoter, carry one promoter element, a -35 box, in one end, whereas the opposite end includes the other promoter component: a -10 box. Formation of the transposon circle brings the -35 and -10 elements together at the circle junction and assembles a strong promoter capable of driving transposase expression from the circle, which facilitates its insertion into a suitable DNA target. Insertion separates the transposon ends, disassembling the strong promoter and rendering initiation of another transposition event dependent on the weak endogenous transposase promoter.

Another regulatory mechanism, called “trans-cleavage,” is considered a “quality control” of the transposition reaction. Transposition requires the formation of a specific complex between the transposase and both transposon ends called a transpososome. In transcleavage regulation, transposase bound at one end is constrained to cleave the opposite transposon end. This obliges prior formation of the transpososome before DNA strand cleavage can occur and ensures that isolated transposon ends are not cleaved serendipitously. This mechanism has been demonstrated biochemically for bacteriophage Mu and for IS50 and is likely to be a common TE regulatory mechanism. Bacteriophage Mu has a relatively elaborate transpososome. Its transposase (MuA) is inactive as a monomer and becomes catalytically proficient only on tetramerization and synapsis between the phage ends. Only two of the four monomers are active. Trans cleavage has also been observed biochemically in Tn5 (IS50) transposition. For IS50, this activity is illustrated clearly by the available crystal structure of the transpososome-DNA complex that represents a post-DNA-cleavage state.

For eukaryotic transposons, assembly of a transpososome is also required for transposition. The Hermes transposase is active as a hexamer on DNA (10) and Himarl transposase is active as a tetramer (11), although it remains unclear whether the Mosl transposase is active as a dimer or a tetramer (12, 13). For the well-described P Element, the transposase is active as a tetramer, and it has been reported recently that GTP acts as an allosteric cofactor for synapsis (14).

Additional regulatory mechanisms can involve various host proteins (i.e., not encoded by the transposon) that are involved in transposome assembly and/or activity. For example, the Escherichia coli histone-like proteins IHF and HU are required for bacteriophage Mu. IHF and HNS, although not required, stimulate Tn10 (IS10) transposition. A more systematic study has revealed several additional host factors that affect transposition of various TE in E. coli either positively or negatively (15). Finally, in the case of the eukaryotic transposon, Sleeping Beauty, the HMG protein is required for integration.

The catalytic domain of this type of transposase is composed of three acidic residues (D, D and E, or D) found in noncontiguous patches and with poor surrounding primary sequence conservation. Structural studies have shown, however, that in all cases investigated (i.e., bacteriophage Mu, IS50, HIV-1, and ASLV integrases, Mos1), DDE motifs fold in a comparable manner. This folding is called the RNaseH fold because it is similar topologically to that found first in RNaseH. It is also found in many other phosphotransferases, such as DNA and RNA polymerases and many nucleases (16). This fold assembles the three conserved residues into an acidic catalytic pocket. The chemical reaction catalyzed by the DDE motif is a hydrolysis that results from nucleophilic attack of the DNA phosphodiester backbone by water. With the appropriate DNA substrate, the target phosphate can be shown to undergo stereochemical inversion in the course of the reaction, which implies a direct single-step, in-line nucleophilic attack (17). It is important to note that no covalent enzyme-DNA intermediate is formed during catalysis by DDE enzymes.

The acidic pocket coordinates two Mg2+ ions jointly between the nucleic acid substrate and the catalytic acidic residues of the enzyme (Fig. 1). This idea was based on the proposition of Steitz (the two-ion model), which concerns the reaction chemistry of exonuclease activity of DNA polymerase (18). This coordination enhances nucleophilic attack of the nucleic acid phospho-sugar backbone and guarantees substrate recognition and catalytic specificity (16).

 

 

Figure 1. Mechanism of DDE enzymes. Only one DNA strand is shown for simplicity. Generally, these reactions take place at each end of the transposon. Transposon sequences are in black, and target sequences are in red. (a) DNA cleavage. The terminal phophodiester bond is shown together with two metal ions (generally but not always Mg2+) T and H. (a1) shows an in-line attack by the water nucleophile (-OH). (a2) shows the pentavalent planar phosphate transition state intermediate in which the two metal ions have approached each other. (a3) shows the cleaved product with the T-bound 3'OH of the transposon end and the leaving phosphate group of the DNA flank. (b) Strand transfer. (b1) shows nucleophilic attack of the target phophodiester bond by the 3'OH (black) of the transferred transposon strand. (b2) shows the pentavalent planar phosphate intermediate with the two metal ions placed closer to each other. (b3) shows the transposon-target joint and the leaving 3'OH group of the target. (c) Hairpin formation and resolution at the transposon end. The water nucleophile and the H and T metal ions are indicated. (d) Hairpin formation at the donor flank. (e) Cleavage of the nontransferred strand within the transposon end. (f) Formation of a forked DNA intermediate.

 

Despite their diversity, DDE transposases catalyze only two chemical reactions: cleavage and strand transfer (Fig. 1a and 1b). Both reactions involve single-strand DNA cleavages. DDE enzymes do not catalyze double-strand cleavage. The cleavage reaction occurs at both ends of the TE, generally using H2O as the nucleophile, to generate 3'-OH ends (19, 20) (Fig. 1a). In the retroviral integrase (IN) proteins, this reaction is known as processing. For several transposons, this reaction is a major regulatory checkpoint because, in these cases, cleavage does not occur on isolated TE ends but requires prior formation of a transposase complex that involves both ends. In several systems, it has been shown that complex assembly evolves through several steps and becomes increasingly stable during this process. This evolution reflects the highly organized architecture of the complex, known as the transpososome (21).

A dynamic model of the catalytic reactions that lead to strand cleavage has been proposed based on structural considerations (22). Assembly is thought to involve coordinate binding of transposase, its DNA substrate(s), and two divalent metal ions. On transposase DNA binding, the two metal ions (H and T for hydrolysis and transfer, respectively; Fig. 1a1) find their appropriate positions in the active site and are poised for catalysis. Metal ion H orients and activates the water molecule (depicted as -OH) for nucleophilic attack. Metal ions T (which is coordinated with an irregular geometry) and H then move closer to each other. The target phosphorus atom adopts a pentavalent transition state (Fig. 1a2), which is then converted into product as the metal ions move back and away from each other (presumably by charge repulsion between T and H). The resulting 5'-phosphate and 3'-OH then dissociate (Fig. 1a3).

In the case of nucleases, the reaction terminates at this step. For transposases, however, the liberated 3'-OH of the transposon end is then used as a nucleophile in a second reaction: trans-esterification. In this reaction, the 3'OH attacks the target DNA, which results in strand transfer or joining of the transposon strand to its target strand. The detailed picture of the DDE cleavage reaction (Fig. 1a) might also be extended to the strand transfer reaction (Fig. 1b). The nucleophile of this reaction would now be the 3'-OH group from the preceding cleavage reaction, which would be coordinated by the T cation (Fig. 1b1) (23). Therefore, the trans-esterification reaction is comparable to that of cleavage but, here, the in-line nucleophlic attack transfers the free 3'-OH end of the transposon in the target DNA (red in Fig. 1b), which creates a transposon-target joint.

Although all transposons with a DDE transposase (“DDE transposons”) use this type of chemistry, a large diversity exists in the overall transposition mechanism. As explained above, DDE transposases catalyze only single-strand cleavage and transfer of the 3'-OH transposon ends (the transferred strand). However, to liberate the transposable element from donor DNA, the transposase must deal with the second DNA strand (also called the nontransferred strand (3, 24). A subclassification of DDE transposons is based on the mechanisms used to manage this.

In the well-characterized bacterial IS4 family (which includes IS3O and ISiO), it is the liberated 3'OH of the transferred strand that is used to attack and to cleave the second strand. This generates a hairpin intermediate (Fig. 1c), in which transferred and nontransferred strands are joined and both DNA strands that flank the ends are removed. Hairpin formation involves a considerable torsion of the DNA, which is aided by extrusion of a subterminal thymine residue from the DNA helix—a flipped-out T (25). This formation has been studied in detail for both ISiO and IS3O and requires a specific group of aromatic residues within the DDE domain. These residues are conserved within the family (26), and they serve to initiate and to stabilize this sequence-specific distortion (27). A second round of cleavage then removes the interstrand hairpin that regenerates the initial 3'OH, which is used subsequently in the final strand transfer reaction.

This type of second strand management is found in eukaryotic transposable elements, such as the hAT group (28), and in V(D)J immunoglobulin-gene rearrangements (2) (Fig. 1d). In these cases, however, hairpin formation occurs on the equivalent of the transposon flanks rather than on the transposon itself. In the case of V(D)J, no specific subterminal T exists, and in the case of the hAT transposons, the hairpin is formed on the transposon flank and can vary between different hAt copies. Thus, hairpin formation in these cases occurs on sequences that are not necessarily conserved, and it seems unlikely that it involves a specific flipped-out T residue.

For the widely dispersed Tc-Mariner transposon group, the transposase first cleaves within the 5' end of the transposon—the nontransferred strand. This activity resembles the nuclease activity (see above) that simply terminates at the cleavage step and does not take in charge the strand transfer step. Moreover, unlike transposition reactions, this cleavage does not require formation of a synaptic complex (29). The transferred strand is cleaved at the very tip of the TE. The fact that the nontransferred strand is cleaved within the transposon results in retention of the few TE-specific bases in the donor molecule after TE excision. After resealing and repair, the donor backbone retains several additional base pairs derived from the TE (called a scar) that marks the passage of the transposon (2).

Interestingly, transposon Tn7 behaves in a similar way but, in this case, the 5' endonuclease activity is supplied by a separate enzyme whose structure resembles that of a type II restriction enzyme (30), and cleavage occurs cleanly at the transposon tip rather than within the TE. Transposition of Tn7, like most bacterial elements, does not leave a scar. In both the Tc-Mariner transposon group and the Tn7 family of transposons, the transposase then cleaves and transfers the 3' end in a true DDE transposition reaction (Fig. 1e).

Finally, cases exist in which the second strand is not processed at all. These include bacteriophage Mu, and the Tn3, IS3, IS3O, and IS236 families. Here, replication is involved intimately in the transposition process itself. In a first step, the transposase cleaves and transfers a transposon end to a target DNA. For phage Mu and Tn3, both transposon ends are transferred directly into target DNA that links both donor and target molecules at each of the transposon ends (2) whereas for IS3 (2), IS3O and IS236 family members, the 3'OH of one transposon end attacks the opposite end. In both cases, a branched structure is generated around which a replication fork is then assembled to resolve this structure (Fig. 1f). In the case of Mu and Tn3, strand transfer is followed by replication and leads to duplication of the transposon. In the case of IS3, IS3O, and IS236 family members, replication leads directly to formation of a transposon circle intermediate and regenerates the original donor locus. The transposon circles, which carry abutted transposon ends, are highly recombinogenic transposition intermediates. They then undergo insertion readily into a suitable target DNA in a reaction that involves concomitant transposon-catalyzed cleavage and concerted transfer of both ends (31).

The Tyrosine and Serine Transposases

Although the reactions catalyzed by the S- and Y- site-specific recombinases are well characterized, little is known about the biochemistry of their cousins, the Y- and S-transposases. These transposases are thought to catalyze strand breakage and transfer as do their site-specific recombinase relatives. Neither of these requires divalent metal ions for catalysis. Although these transposases show site-specificity for the ends of the donor transposon, they seem more flexible than classic site-specific recombinases in the DNA target sequences they recognize and use as the partner during integration. Strand cleavage catalyzed by the Y- and S-recombinase enzymes occurs using a tyrosine or serine hydroxyl group, respectively. This generates a phosphotyrosine or phosphoserine intermediate. The Y-recombinases form a 3' phosphotyrosine bond, whereas the S-recombinases create a 5' phosphoserine bond. Various host accessory proteins are required in these reactions to generate protein-DNA complexes with the correct architecture for catalysis.

The Y2- Rep and Relaxase proteins are less well understood, and the Y2 IS91 transposase is proving to be difficult to work with. Again, it is assumed largely that the Y2 enzyme behaves the same as the related plasmid and phage Rep and plasmid Relaxase proteins. The mechanism of the final group, the Y1 transposases, is now relatively well understood.

S-recombinases

Cleavage and strand transfer are mediated by successive transesterification reactions within a synaptic complex that includes the two partner DNA sequences (called core sequences) bound by a recombinase tetramer. In the transposases, the DNA sequences would be the transposon ends. The nucleophile hydroxyl groups of the catalytic serine residues initiate recombination by attacking specific phosphodiester bonds (the scissile phosphates) of the DNA backbone (Fig. 2a1). Cleavage results in covalent attachment of the proteins to the 5' ends of the cleaved DNA strands and production of 3'-OH free ends (Fig. 2a2). S enzymes cleave the four strands of the paired core sites concomitantly at both edges of a 2 bp central region. This generates an intermediate that contains 2 bp staggered DNA ends held together by interactions between the bound recombinase tetramer (Fig. 2a2 and 2a3). An exchange of subunits occurs, which implies a rotation of 180° between the two pairs of half sites (Fig. 2a3). Strand rotation has been modeled as a rotation of the subunits that use a shared hydrophobic intersubunit surface (32). This rotation is followed by strand transfer via nucleophilic attack of the covalent DNA-enzyme bonds by the 3' hydroxyl ends generated by cleavage (Fig. 2a4). The two pairs of strands are exchanged concomitantly to restore strand continuity (Fig. 2a5).

S-transposons fall into two distinct classes. Some are conjugative transposons (CTns) found in Clostridia species and use “large S enzymes” for excision and insertion. These TEs also carry genes and DNA sites involved in their conjugative transfer from one bacterial cell to another. Of these, Tn5397 and Tn5398 are large self-transferable CTns, whereas Tn4451 and Tn4453 are shorter but can be mobilized by other elements. These ISs encode atypical S enzymes with a reverse order of DNA binding and catalytic domains compared with S-recombinase cousins. The second class includes IS507 of Helicobacter pylori (33) for which no published biochemical information exists.

 

Figure 2. Proposed mechanism of Y- and S-transposases. (a) The S-transposases. Proteins are shown in yellow and transposon ends in green. This figure is based on the known mechanism of serine site-specific recombinases. The serine nucleophiles are indicated (S) as are the 3'OH liberated on formation of the phophoserine bonds. (a1) Synapsis and attack by the serine nucleophile. (a2) Formation of 5' phophoserine enzyme substrate intermediate and liberation of the 3'OH. (a3) Rotation of the substrates (large arrow). (a4) Realignment and 3'OH attack of the phophoserine bonds. (a5) Resealing the broken strands generates a transposon circle and a reclosed donor backbone from which the transposon has been deleted. (b) The Y-transposases. Proteins are shown in yellow and transposon ends in green. This figure is based on the known mechanism of tyrosine site-specific recombinases. The tyrosine nucleophiles are indicated (Y) as are the 5'OH liberated on formation of the phophotyrosine bonds. (b1) Synapsis and attack by the tyrosine. The top right and bottom left subunits are active for cleavage. (b2) Cleavage liberates the 3'OH, which then attacks the phosphotyrosine bond on the opposite (horizontal as drawn) subunit to reseal the DNA strand in a first strand transfer step. (b3) The first strand transfer product. (b4) Second strand cleavage. The top left and bottom right transposase subunits are activated and cleave the opposite strands. (b5) Second strand transfer results in formation of a transposon joint and a donor backbone from which the transposon has been deleted as a transposon circle. (c) The Y1 transposases. The left and right transposon ends are shown in red and blue, respectively. (c1) Diagram that shows the potential secondary structures at the left and right ends. (c2) TnpA binding and cleavage. The top strand is recognized by TnpA (yellow ellipses) that catalyzes cleavage via a 5’-phosphotyrosine linkage (black arrows). This cartoon shows that the covalent bond is formed at the 5' end of the left end and at the 5' end of donor flank at the right end. (c3) Cleavage is then followed by exchange between the left and right ends to generate a Right End-Left End transposon joint and to liberate a single strand transposon circle. (c4) Excision of IS608 and closure of the flanking backbone would leave a deletion bubble in the bottom strand. (c5, c6) Replication of the donor plasmid (arrow) would generate a copy of the original donor plasmid with an intact, double strand IS608 copy (c6), and a copy of the plasmid devoid of the IS (c5).

 

Y-recombinases

Recombination is catalyzed by a recombinase tetramer bound at the two partner core sequences that are brought together in an antiparallel configuration. Only two opposing monomers at a time are in a configuration competent for cleavage (Fig. 2b1). The first pair of strands is cleaved in a concerted manner at one edge of the central region by a nucleophilic attack of the hydroxyl group of the conserved tyrosine (Fig. 2b1 and 2b2). It generates a 3' phosphotyrosyl bond with the cleaved strand that lliberats a corresponding 5’-OH (Fig. 2b2). The polarity of attack and cleavage is opposite to that found with S-recombinases. The 5’OH then attacks the phosphotyrosyl bond created during cleavage in the partner core sequence to produce a four-way “Holliday” junction intermediate (Fig. 2b3). The first cleavage and/or strand exchange allows the reaction to proceed to the second pair of strand exchanges that occurs at the other end of the central region of the core site (Fig. 2b4) to generate the final product (Fig. 2b5). Exchange of the second pair of strands is separated temporally and spatially from the first. This exchange, together with the inverted polarity of cleavage, represents the major differences to S-recombinase driven catalysis.

Y transposons are heterogeneous. They share the capacity to be transferred by a conjugative mechanism from cell to cell and are referred to as conjugative transposons (CTns) (2). Often, they carry antibiotic resistance determinants (usually the tetM gene) and are important vectors for disseminating this antibiotic resistance. Some are self transferable (e.g., Tn915 from Streptococci and Tn1525 from Enterococci), whereas others are mobilizable by functions provided in trans by other elements such as conjugative plasmids or other CTns (e.g., the NBUs elements from Bacteroides sp.). They can have a very broad host range and seem to contribute significantly to gene transfer in complex bacterial populations.

Y2-transposases

These enzymes are members of a family of nucleases (34) known as the HUH superfamily because they include a conserved histidine-hydrophobic-histidine motif that provides two of three ligands to an essential divalent metal ion cofactor. Generally, these enzymes are monomeric. Members include proteins that initiate conjugative plasmid transfer from cell to cell or catalyze rolling circle replication (RCR) in certain bacteriophages and plasmids. Prokaryotic members include the IS91 family and the newly identified ISCR group (35), whereas eukaryotic members include the helitrons (22).

The similarity between RCR and rolling circle transposition (RCT) is underlined by the fact that the “left end” of IS91 resembles a rolling circle replication origin—a structured region that is recognized and undergoes single strand-specific cleavage to initiate replication. The enzymes carry five conserved blocks of amino acids, one of which includes a pair of tyrosine residues involved in catalysis. The best-characterized reactions that use Y2 enzymes are those involved in phage replication (e.g., фX174) They use two active site tyrosines and cleave the DNA by releasing the 3' OH after 5’ phosphotyrosine formation. One difference between RCR and RCT is that, in the case of RCT, strand transfer from donor to target molecule must occur. Several possible ways exist in which this could be integrated into this type of process but the exact mechanism has yet to be determined at the biochemical level.

The RCT mechanism confers several interesting features on IS91-family members. These elements do not carry terminal inverted repeats nor do they generate target site duplications as do the DDE transposons. IS91 itself inserts with a specific orientation at the 3' end of a conserved tetranucleotide sequence (5'CTTG3' or 5'GTTC3'), which is probably involved in transposition initiation and termination and also required for additional efficient transposition. Deletion of the downstream (“right”) end results in “one-ended” transposition in which different lengths of vector DNA neighboring the deletion accompany the element to its new target site. These terminate with a 5'CTTG3' or 5'GTTC3' tetranucleotide located in the vector (2).

Y1-transposases

These enzymes were identified and characterized more recently (6). They are also part of the HUH superfamily of nucleases but currently characterized members have only one catalytic tyrosine and form obligatory dimers (7, 8, 36). These transposases are less than half the size of many other transposases.

The best-characterized example is that of the Helicobacter pylori element, IS60S, although this IS group is extremely widespread and has representatives in both the bacteria and the archaea (37). IS60S does not possess terminal IRs. The left (LE) and right ends (RE) include palindromic repeats, which form DNA hairpins located at some distance from the cleavage sites. The cleavage site at the left end is 19b upstream from the foot of the LE hairpin and at the right end it is 9b downstream from the RE hairpin (Fig. 2c1). IS60S insertion occurs 3' to a tetranucleotide TTAC conserved in the flanking DNA, which abuts LE (38) directly (Fig. 2c1). Moreover, it is also required for subsequent transposition (7). IS60S transposition is strand specific. It occurs by precise excision of the “top” transposon strand to generate a circular intermediate with abutted copies of RE and LE (the transposon joint or RE-LE junction; Fig. 2c3 and reclosure of the flanking DNA, which preserves the target TTAC (7).

TnpA behaves as a dimer in solution and a crystal structure indicated that the molecule forms an elongated and flat dimer (8). The crystal structure of the complex formed by TnpA and a 22 nt long, single-stranded oligonucleotide that represents the RE palindrome showed a DNA hairpin (an imperfect palindrome) bound to each of the two recognition sites in the TnpA dimer (8).

TnpA binds single strand (ss) DNA that carries either “top strand” LE or RE (see Fig. 2c2) much more strongly than double strand (ds) DNA ends. It did not bind the ss “bottom strand.” TnpA catalyzes efficient strand- and sequence-specific cleavage of a single “top” strand oligonucleotides, which includes LE or RE. IS60S transposition necessitates formation of an enzyme-substrate intermediate in which TnpA is attached covalently at a DNA end via a 5' phosphotyrosine bond. Because of the conserved polarity of cleavages required at the ends, TnpA is joined to the 5' end of the transposon at LE but to the 5' end of the flanking donor DNA at RE (Fig. 2c2) (8). Nucleophilic attack of the LE phosphotyrosine bond by the free RE 3'OH results in reclosure of the ss transposon to form a circle (Fig. 2c3), whereas attack of the RE flank phosphotyrosine bond by the LE flank 3'OH reseals the donor backbone (Fig. 2c4). The transpo- son circle that carries an RE-LE junction can undergo insertion into a suitable TTAC target. The resulting donor molecule intermediate (Fig. 2c4) could be resolved by replication to generate a copy of the original donor plasmid molecule (Fig. 2c6) and a copy from the deleted transposon (Fig. 2c5).

Conclusions

Transposition is a general term that covers a variety of different TEs and includes several diverse chemistries. It should be underlined that although several partial structures of DDE enzymes have been determined, the structure of only a single complete DDE transposase has been obtained complexed with appropriate ends. For other types of transposase, two structures of Y1 transposases are available (8, 36), one of which includes its DNA substrate (25). Additional structural information will be critical to understand how these fascinating enzymes function. The information presented above is derived largely from in vitro experiments, although a a body of in vivo information also exists. One major challenge in the field of transposition at present is to determine how these elements interact with and are regulated by their respective hosts.

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Further Reading

Craig NL, et al. Mobile DNA II. 2002. American Society of Microbiology, Washington, DC.