Mechanism of Single‐Stranded DNA Transposition: A Structural Perspective

Abstract

Transposons are mobile genetic elements that have shaped evolution and thus constitute much of modern genomes. Their movements cause diseases, change regulatory patterns and spread antibiotic resistance. Single‐stranded deoxyribonucleic acid (ssDNA) transposition, is carried out by a unique transposase protein that recognises a hairpin structure formed on ssDNA, excises the transposon as a single‐stranded circle, and integrates it specifically into ssDNA target. The use of ssDNA alleviates the cumbersome processing of the second DNA strand and implies a built‐in regulation of transposition via substrate limitation in vivo. Moreover, it allows selection of a specific sequence for integration via base‐pairing between transposon and target DNA. Due to this target site recognition strategy the transposon can be redirected to novel sites and may permit the development of programmable site‐specific genetic tools for research and gene therapy. Furthermore, unexpected similarities suggest that ssDNA transposons share mechanistic features with repetitive extragenic palindromes and ribonucleic acid (RNA) machines.

Key Concepts:

  • Transposons are mobile genetic parasites that can move from one location in the genome to another.

  • Transposons are abundant in most prokaryotic and eukaryotic organisms, where they contribute to genome structure, function and evolution.

  • Single‐stranded DNA (ssDNA) transposition is carried out solely on ssDNA substrates and intermediates.

  • ssDNA transposons alleviate the need for cutting and pasting two DNA strands and thereby preserve chemical energy.

  • The transposon encodes a transposase protein, which recognises, cleaves and integrates the two ends of the transposon in the context of a synaptic complex called the transpososome.

  • The ssDNA transpososome recognises its target site sequence specifically via base‐pairing interactions between a part of the transposon DNA and the cleavage site.

  • In vivo, ssDNA transposition is limited by the availability of ssDNA, which provides a built‐in regulatory mechanism.

  • Their mechanism of target site recognition allows ssDNA transposons to be redirected to novel target site sequences, potentially allowing the development of unique genetic tools for research and medicine.

  • Repetitive extragenic palindromes (REPs) may have derived from ssDNA transposons.

  • In complex with a helper protein, ssDNA can form complex intertwined tertiary structures providing unique functions that were previously seen only for RNA.

Keywords: single‐stranded DNA; DNA transposition; crystal structure; molecular mechanism; genetic tools

Figure 1.

DNA transposition. (a) The general pathway of DNA transposition. The transposon is shown in yellow, with its conserved end sequences (typically terminal inverted repeats) in black. Following expression of the transposase (green ball), it binds to the two transposon ends and brings them together forming the transpososome. In the transpososome, DNA cleavages take place at both transposon ends excising the element from its original ‘donor’ location (blue). The donor is left broken until it is repaired by host proteins. The trasnpososome can recruit a target DNA (orange), cleave both strands of the target and integrate the element to the new location. (b) Alternative transposition pathways. The upper panel shows schematic representations of all transposition pathways described to date, highlighting known characteristic transposition intermediates. In the lower panel the nature of the nucleophiles used and the mechanism of second strand processing are described for each pathway. This Figure is courtesy of Drs. Alison Burgess Hickman and Fred Dyda.

Figure 2.

The IS608 transposon. (a) Architecture of the IS608 transposon. Nucleotide sequence and secondary structure are shown. LE is shown in red, RE in blue, with the 4 nt 5′ extensions (‘guide’ sequence) in yellow or cyan, respectively. Donor/target DNA is in black. The colour code is conserved throughout this article. Bases not fully conserved among IS608 isolates from various H. pylori strains are shown as lower case letters. Note that the transposon end sequences are asymmetric: (1) there is no sequence similarity between the two termini; (2) the RE IP is 10 nt away from the right end of the element, whereas the LE IP is 19 nt from the LE; (3) the linker between RE and RE IP is strictly conserved, whereas it is more variable on the LE; and (4) the donor sequence flanking LE is strictly conserved (the target site), whereas there is no conservation in the RE flank. (b) Crystal structure of the transposase in complex with the LE IP and the cleavage site (PDB ID 2vjv). (c) Crystal structure of the transposase in complex with the transposon RE (PDB ID 2vju). The TnpA dimer (sand and light blue) binds two IP hairpins. The catalytic residues are highlighted in ball‐and‐stick representation and labelled. Red circles point out the locations of the two Gly residues that provide the pivot points for αD movement.

Figure 3.

Mechanism of ssDNA transposition. Upon LE and RE binding by the transposase (sand and light blue), Y127 of each monomer (marked as yellow and green stars) cleaves the transposon ends and becomes covalently attached to the 5′ side of the gap (‘cleavage’). Movement of the αD helices from trans to cis arrangement (‘conformational switch’) and resolution of the phosphotyrosine intermediates creates a transposon junction and a sealed donor backbone (‘strand transfer’). This completes transposon excision (black arrows). For integration (grey arrows), the donor backbone is replaced by target DNA containing TTAC, which is recognised by the LE. Cleavage, movement of the αD helices, and resolution of the phosphotyrosine intermediates (‘ligation’) results in transposon insertion into target DNA immediately 3′ of TTAC.

Figure 4.

Cleavage site recognition in the IS608 (a, PDB ID 2vjv) and ISDra2 (b, PDB ID 2xm3) transpososomes. In the structural figures (left panels), the transposase protein is shown in space filling surface representation for clarity and its nucleophile tyrosine is marked by green asterisk. In IS608, the 4 nt preceding the cleavage site are recognised via base‐pairing interactions with a segment of the transposon LE (‘guide’ sequence). Base‐pairing interactions are shown schematically in the right side panels. In ISDra2, a specific binding pocket on the protein recognises the 5th nucleotide in the cleavage site (green circle). (c) The intertwined structure of IS608RE. At the base of the hairpin two base triples are formed, which are marked by dashed lines representing H‐bonding interactions.

Figure 5.

Recognition and discrimination of the transposon ‘top’ strand. Comparison of the binding of the hairpin loop by IS608 TnpA (left) and TnpADra2 (right). The distinguishing features of the hairpins are shown in smudge green: the T at the hairpin tip (a) and the unpaired T in the stem (c) of the IS608 hairpin, and the T at the hairpin tip (b) and the mismatched G:T pair (d) of the ISDra2 hairpin. A web of hydrogen‐bonding interactions and a bound Mg2+‐ion mediate the interaction between Arg14 and the mismatch on the ISDra2LE.

Figure 6.

Strand bias of IS200/IS605 family transposition in vivo. Schematic representation of replication forks progressing through an IS200/IS605 family transposon in the two possible directions. Note that if the fork passes such that the active transposon ‘top’ strand is located on the leading strand template the transposon cannot be excised. In turn, if the origin of replication is inverted, the tranposon ‘top’ strand will be on the lagging strand template and can be mobilised.

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How to Cite close
Barabas, Orsolya(Jul 2012) Mechanism of Single‐Stranded DNA Transposition: A Structural Perspective. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0023178]