Model of transposon excision by members of the IS200/IS605 family of insertion sequences (ISs) and sequence alignment of some of their transposases. (A) The IS is shown as a thin black line with its left end (LE) in red and right end (RE) in blue. The imperfect palindromes (IP) are shown schematically as hairpins, which are flanked on their 5′ sides by solid boxes representing the four nucleotides involved in cleavage (or target) site recognition. The transposase, TnpA, is shown as a dimer with orange subunits. Step 1: TnpA synapses the two IS ends by binding the IPs, and brings the cleavage sites (shown as white boxes) at the IS ends into the active sites through DNA–DNA interactions between the nucleotides 5′ of each end (TTGAT at LE, TTCAA at RE in the case of ISDra2) and those at the 5′ sides of the IPs. For clarity, the two IS ends are shown binding in the same orientation to each monomer of the dimer; however, in the determined structures, one IP is rotated by 180° relative to the other (vide infra). Step 2: End cleavage mediated by the active site Tyr on helix αD results in covalent attachment (represented by a red hexagon) of one TnpA subunit to the cleaved LE and the other subunit to the flanking DNA 3′ of the cleaved RE. Step 3: A trans-to-cis movement of the αD helices brings the two ends of the donor DNA together in one active site (monomer on left) and the LE into the proximity of the cleaved RE (monomer on right). Step 4: Nucleophilic attack of the two 3′-OH groups onto the covalent phosphotyrosine intermediates results in sealed donor DNA and a circular transposon intermediate. Reversal of these steps (grey arrows) represents insertion of the transposon intermediate into target DNA. (B) Sequence alignment of the transposases of ISDra2, the IS represented by PDB code 2fyx, and IS608 superimposed on the secondary structure (β-strands in blue, α-helices in red) of IS608 TnpA. The amino acid numbering is for ISDra2 transposase. The active site His (on the fourth β-strand) and Tyr residues (on helix αD) are in bold, and residues putatively involved in the trans-to-cis transition in blue.

Structures of TnpA_Dra2 bound to its transposon ends. (A) ISDra2 LE and RE DNA sequences. The overall structures of the TnpA_Dra2 dimer bound to (B) the LE IP (in red and orange) and a short ssDNA (in black) representing the five nucleotides 5′ of the LE cleavage site, which is equivalent to the target site, and (C) the RE IP (in light and dark blue) extended to the transposon 3′ end. The DNA colour scheme corresponds to that shown in Figures 1A and 2A. Residues of TnpA_Dra2 highlighted in green are specifically referred to in the text (Y30, H67, H69, W107, and Y132).

DNA binding by TnpA_Dra2: G:T mismatch recognition and discrimination between the top and bottom strand of the transposon. (A) Comparison of the binding of the tip of the hairpin loop by IS608 TnpA (left; PDB code 2A6O) and TnpA_Dra2 (right). The distinguishing features of the hairpins are shown in shades of blue: the unpaired T in the stem of the IS608 hairpin and the T at the hairpin tip (left), and the mismatched G:T base pair and the T at the hairpin tip of the ISDra2 hairpin (right). (B) Space-filling representation of the complex between TnpA_Dra2 (two subunits shown in shades of green) and LE27 (in orange) highlighting the position of Arg14. (C) A web of hydrogen-bonding interactions and a bound Mg²⁺ ion mediate the interaction between Arg14 and the G+20:T+33 mismatch on the LE. (D) Dissociation constants for binding between the IS608 and ISDra2 transposases and their IPs and variants determined by fluorescence anisotropy measurements.

Transposon end and cleavage site recognition by TnpA_Dra2. On the left are the overall TnpA_Dra2 dimer structures corresponding to the complexes TnpA/LE27/T5 (A) and TnpA/RE34 (B). In the middle are close-up views of the active sites. The cartoons on the right show the state during the transposition pathway captured by the crystal structures, corresponding to the model in Figure 1A. In these cartoons, the vertical black arrows correspond to the sites of cleavage and the pattern of base-pairing interactions at the cleavage sites at LE (A) and RE (B) are indicated by the black lines. The inset box shows the corresponding pattern of base-pairing interactions for IS608 (Barabas et al, 2008). The dashed lines indicate portions of DNA not present in the structures.

The TnpA_Dra2 active site is fully assembled in the presence of Cd²⁺ but is distorted by Zn²⁺. (A) The structure of the complex of TnpA_Dra2 Y132F in the presence of Cd²⁺ (white sphere) bound to LE27 (not visible) and an oligonucleotide, T5↓G, that traverses the LE cleavage site where target nucleotides are shown in black and the first 5′ transposon nucleotide in red. (B) TnpA_Dra2 is fully active for LE cleavage in the presence of Cd²⁺ but not Zn²⁺. Cleavage of a 13-mer target sequence results in a covalent complex between the product and TnpA_Dra2, and is detected by Coomassie-stained SDS–PAGE as the formation of a higher molecular weight band. (C) The active site of TnpA_Dra2 is distorted in the presence of Zn²⁺ (blue sphere), explaining the lack of cleavage activity.