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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. Interest ingly, 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.
- Top of page
- Materials and methods
The composite bacterial transposon Tn10 transposes by a non-replicative mechanism. The complete transposon, or one of its two flanking IS10 modules, is first excised by a pair of double strand breaks and then inserted into a new target site. These steps are carried out by the Tn10-encoded transposase protein, a 46 kDa protein that binds to specialized DNA sequences located at transposon termini or ‘ends’. Two transposase monomers bind per transposition module (Kennedy, 1999) and orchestrate the assembly of a higher order protein–DNA complex called a transpososome, in which the two transposon ends are held together. All of the chemical steps take place in the context of the transpososome (Kleckner et al., 1996). A particularly fascinating aspect of the Tn10/IS10 transposition reaction is that the single active site in the transposase protein is used repeatedly to catalyze the four different chemical steps that occur at each transposon end (Figure 1). In previous work, we have defined the orientation of different DNA strands in the single active site for each of the four chemical steps (Kennedy et al., 2000). However, a more detailed understanding of each of these steps requires definition of the transposase–DNA interactions that govern them. This remains an important goal in this and other related transposition reactions.
Figure 1. Structure of IS10 Right and transposase-mediated chemical steps in Tn10/IS10 transposition. IS10 Right (thick lines) encodes transposase protein and contains the binding determinants for transposase (filled rectangles) at its termini, the ‘inside’ (IE) and ‘outside’ (OE) end sequences. The inverted repeat sequence (half arrows) contains the primary specificity determinants for transposase binding. In addition, the OE also contains a binding site for Escherichia coli IHF (open rectangle) and a second ‘distal’ transposase-binding site. The four chemical steps that occur at each transposon end are indicated; thin lines indicate flanking donor DNA. The top strand shown is the ‘non-transferred’ strand (NTS) and the bottom strand is the ‘transferred’ strand (TS).
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As shown in Figure 1, Tn10 excision takes place through a hairpin mechanism (Kennedy et al., 1998). The formation of a double strand break in DNA by a hairpin mechanism also occurs in V(D)J recombination and Tn5 transposition, and is likely for a number of plant transposons that utilize the cut-and-paste mode of DNA transposition (Coen et al., 1989; Fedoroff, 1989; McBlane et al., 1995; Colot et al., 1998; Bhasin et al., 1999). In the Tn10, Tn5 and V(D)J systems, hairpin formation involves attack of a 3′ OH DNA strand terminus on a phosphate group situated directly across the double helix from the site of the initial nick, giving a perfectly self-complementary DNA hairpin (McBlane et al., 1995; Kennedy et al., 1998; Bhasin et al., 1999). Also, in the V(D)J and Tn10 systems, it has been shown that this reaction takes place by a direct in-line attack mechanism wherein hairpin formation is contemporaneous with the complete release of transposon from flanking DNA sequences (van Gent et al., 1996; Kennedy et al., 2000).
Hairpin formation is considered to be a relatively difficult reaction because the functional groups involved in the reaction chemistry are separated by 16–18 Å in B-form DNA. It is therefore expected that this step would be associated with a significant distortion in the DNA structure at the site of the reaction chemistry. The first evidence of this came from studies in the V(D)J system where it was shown that the introduction of base pair mismatches immediately adjacent to the site of hairpin formation relieved the inhibitory effect of suboptimal flanking DNA sequences on hairpin formation (Cuomo et al., 1996; Ramsden et al., 1996). More recently, the structure of a Tn5 transpososome thought to represent a stage of the reaction following cleavage of the transposon from the donor DNA has been solved. In this complex, the terminal two base pairs of the transposon end are unpaired and base stacking interactions in this region are disrupted by the ‘flipping’ of one of the bases at the second position out of the double helix (Davies et al., 2000). Details of the molecular events that govern hairpin formation have not been addressed previously in the Tn10 system and are the main focus of the current study.
Tn10 transposase interactions with its end sequences have been studied extensively by DNA footprinting and interference approaches as well as by functional studies on mutant forms of the end sequences (Huisman et al., 1989; Haniford and Kleckner, 1994; Sakai et al., 1995, 2000; Kleckner et al., 1996; Sakai and Kleckner, 1996). The picture that emerges from these studies is that the primary determinants for end recognition are located in the interior portion of the inverted repeat sequence, including bp 6–13. DNA sequence information is also important at the tip of the inverted repeat as mutations at bp 1–3 strongly inhibit transposition. However, the effects of these mutations are manifested at a stage subsequent to the initial binding of transposase to the end sequence. Given that previous mutational studies were carried out prior to the identification of the hairpin intermediate in the Tn10 system, and considering the complexity of this reaction, the possibility arises that the terminal base pair mutations may interfere with hairpin formation.
A similar transposon end organization to that described above has also been demonstrated in the Tn5 and IS903 systems (Derbyshire et al., 1987; Jilk et al., 1996; Davies et al., 2000). Interestingly, the terminal four base pairs of the outside ends of Tn10 and Tn5 are identical in sequence. Given that both systems employ the hairpin mechanism, this could reflect similar structural requirements for hairpin formation in the two systems. At the protein level, the Tn10 and Tn5 transposases exhibit only 20% amino acid sequence identity. However, sequence similarity is extensive throughout the entire lengths of these two proteins, with the exception of a block of 44 amino acids present at the C-terminus of the Tn5 transposase. The most highly conserved block of amino acids in the two proteins is a region that contains the E of the DDE catalytic triad. This includes the Y-2-R-3-E-6-K signature, a hallmark of the IS4 family of transposons, of which both Tn10 and Tn5 are members (Mahillon et al., 1985). The structure of the Tn5 synaptic complex has provided possible clues towards defining amino acid residues that play an important role in hairpin formation (Davies et al., 2000). For example, W298 and Y237 stack against the flipped out base at position 2, and Y319 forms a hydrogen bond with a phosphate oxygen at the site of a sharp kink between residues 1 and 2. Such interactions are presumably important in positioning the 5′ terminus of the non-transferred strand in close proximity to the 3′ OH of the transferred strand. This information provides a framework for defining, through mutational analysis, amino acids in Tn10 transposase that may also function in hairpin formation. At the present time, only one residue in Tn10 transposase, P167, has been shown to play an important role in this step (Kennedy et al., 1998). More generally, such studies will be important in addressing how closely related the structures of the two proteins are.
In the current work, we have examined the effects of a selected group of transposon end mutations and amino acid substitutions in transposase on Tn10 excision. We define the specific stages at which these changes affect Tn10 excision and relate these changes to the interactions that take place between transposase and the tip of the outside end.