Mechanism of Single‐Stranded DNA Transposition: A Structural Perspective


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.



Barabas O, Ronning DR, Guynet C et al. (2008) Mechanism of IS200/IS605 family DNA transposases: activation and transposon‐directed target site selection. Cell 132(2): 208–220.

Beese LS and Steitz TA (1991) Structural basis for the 3′‐5′ exonuclease activity of E. coli DNA polymerase I: a two metal ion mechanism. EMBO Journal 10(1): 25–33.

Bertels F and Rainey PB (2011) Within‐genome evolution of REPINs: a new family of miniature mobile DNA in bacteria. PLoS Genetics 7(6): e1002132.

Chalker DL and Yao MC (2011) DNA elimination in ciliates: transposon domestication and genome surveillance. Annual Review of Genetics 45: 227–246.

Chen JM, Stenson PD, Cooper DN and Ferec C (2005) A systematic analysis of LINE‐1 endonuclease‐dependent retrotranspositional events causing human genetic disease. Human Genetics 117(5): 411–427.

Craig NL (2001) Transposases and Integrases. eLS. Chichester: John Wiley & Sons Ltd.

Curcio MJ and Derbyshire KM (2003) The outs and ins of transposition: from mu to kangaroo. Nature Reviews Molecular Cell Biology 4(11): 865–877.

Grindley ND, Whiteson KL and Rice PA (2006) Mechanisms of site‐specific recombination. Annual Review of Biochemistry 75: 567–605.

Gueguen E, Rousseau P, Duval‐Valentin G and Chandler M (2005) The transpososome: control of transposition at the level of catalysis. Trends in Microbiology 13(11): 543–549.

Guynet C, Achard A, Ton‐Hoang B et al. (2009) Resetting the site: redirecting integration of an insertion sequence in a predictable way. Molecular Cell 34(5): 612–619.

Guynet C, Hickman AB, Barabas O et al. (2008) In vitro reconstitution of a single‐stranded transposition mechanism of IS608. Molecular Cell 29(3): 302–312.

Han JS and Boeke JD (2005) LINE‐1 retrotransposons: modulators of quantity and quality of mammalian gene expression? Bioessays 27(8): 775–784.

He S, Hickman AB, Dyda F et al. (2011) Reconstitution of a functional IS608 single‐strand transpososome: role of non‐canonical base pairing. Nucleic Acids Research 39(19): 8503–8512.

Hickman AB, Chandler M and Dyda F (2010a) Integrating prokaryotes and eukaryotes: DNA transposases in light of structure. Critical Reviews in Biochemistry and Molecular Biology 45(1): 50–69.

Hickman AB, James JA, Barabas O et al. (2010b) DNA recognition and the precleavage state during single‐stranded DNA transposition in D. radiodurans. EMBO Journal 29(22): 3840–3852.

Ivics Z, Li MA, Mates L et al. (2009) Transposon‐mediated genome manipulation in vertebrates. Nature Methods 6(6): 415–422.

Karberg M, Guo H, Zhong J et al. (2001) Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nature Biotechnology 19(12): 1162–1167.

Kennedy AK, Haniford DB and Mizuuchi K (2000) Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity. Cell 101(3): 295–305.

Kersulyte D, Velapatino B, Dailide G et al. (2002) Transposable element ISHp608 of Helicobacter pylori: nonrandom geographic distribution, functional organization, and insertion specificity. Journal of Bacteriology 184(4): 992–1002.

Khan SA (1997) Rolling‐circle replication of bacterial plasmids. Microbiology and Molecular Biology Reviews 61(4): 442–455.

Koonin EV and Ilyina TV (1993) Computer‐assisted dissection of rolling circle DNA replication. Biosystems 30(1–3): 241–268.

Kulkosky J, Jones KS, Katz RA, Mack JP and Skalka AM (1992) Residues critical for retroviral integrative recombination in a region that is highly conserved among retroviral/retrotransposon integrases and bacterial insertion sequence transposases. Molecular and Cellular Biology 12(5): 2331–2338.

Lambowitz AM and Zimmerly S (2004) Mobile group II introns. Annual Review of Genetics 38: 1–35.

Mahillon J and Chandler M (1998) Insertion sequences. Microbiology and molecular biology reviews. Microbiology and Molecular Biology Reviews 62(3): 725–774.

McClintock B (1950) The origin and behavior of mutable loci in maize. Proceedings of the National Academy of Sciences of the USA 36(6): 344–355.

Mennecier S, Servant P, Coste G, Bailone A and Sommer S (2006) Mutagenesis via IS transposition in Deinococcus radiodurans. Molecular Microbiology 59(1): 317–325.

Mizuuchi K (1992) Transpositional recombination: mechanistic insights from studies of mu and other elements. Annual Review of Biochemistry 61: 1011–1051.

Noller HF (2005) RNA structure: reading the ribosome. Science 309(5740): 1508–1514.

Nowotny M, Gaidamakov SA, Crouch RJ and Yang W (2005) Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal‐dependent catalysis. Cell 121(7): 1005–1016.

Nunvar J, Huckova T and Licha I (2010) Identification and characterization of repetitive extragenic palindromes (REP)‐associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes. BMC Genomics 11: 44.

Parks AR and Peters JE (2009) Tn7 elements: engendering diversity from chromosomes to episomes. Plasmid 61(1): 1–14.

Pasternak C, Ton‐Hoang B, Coste G et al. (2010) Irradiation‐induced Deinococcus radiodurans genome fragmentation triggers transposition of a single resident insertion sequence. PLoS Genetics 6(1): e1000799.

del Pilar Garcillan‐Barcia M, Bernales I, Mendiola MV and de la Cruz F (2001) Single‐stranded DNA intermediates in IS91 rolling‐circle transposition. Molecular Microbiology 39(2): 494–501.

Plasterk RH, Izsvak Z and Ivics Z (1999) Resident aliens: the Tc1/mariner superfamily of transposable elements. Trends in Genetics 15(8): 326–332.

Rocco F, De Gregorio E and Di Nocera PP (2010) A giant family of short palindromic sequences in Stenotrophomonas maltophilia. FEMS Microbiology Letters 308(2): 185–192.

Ronning DR, Guynet C, Ton‐Hoang B et al. (2005) Active site sharing and subterminal hairpin recognition in a new class of DNA transposases. Molecular Cell 20(1): 143–154.

Ruf S, Symmons O, Uslu VV et al. (2011) Large‐scale analysis of the regulatory architecture of the mouse genome with a transposon‐associated sensor. Nature Genetics 43(4): 379–386.

Siguier P, Perochon J, Lestrade L, Mahillon J and Chandler M (2006) ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Research 34(Database issue): D32–D36.

Thornburg BG, Gotea V and Makalowski W (2006) Transposable elements as a significant source of transcription regulating signals. Gene 365: 104–110.

Ton‐Hoang B, Guynet C, Ronning DR et al. (2005) Transposition of ISHp608, member of an unusual family of bacterial insertion sequences. EMBO Journal 24(18): 3325–3338.

Ton‐Hoang B, Pasternak C, Siguier P et al. (2010) Single‐stranded DNA transposition is coupled to host replication. Cell 142(3): 398–408.

Ton‐Hoang B, Siguier P, Quentin Y et al. (2012) Structuring the bacterial genome: Y1‐transposases associated with REP‐BIME sequencesdagger. Nucleic Acids Research 40(8): 3596–3609.

Toor N, Keating KS, Taylor SD and Pyle AM (2008) Crystal structure of a self‐spliced group II intron. Science 320(5872): 77–82.

Volff JN (2006) Turning junk into gold: domestication of transposable elements and the creation of new genes in eukaryotes. BioEssays: News and Reviews in Molecular, Cellular and Developmental Biology 28(9): 913–922.

Williams DA (2008) Sleeping beauty vector system moves toward human trials in the United States. Molecular Therapy: Journal of the American Society of Gene Therapy 16(9): 1515–1516.

Woltjen K, Michael IP, Mohseni P et al. (2009) piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature 458: 766–770.

Zahradka K, Slade D, Bailone A et al. (2006) Reassembly of shattered chromosomes in Deinococcus radiodurans. Nature 443(7111): 569–573.

Zhou L, Mitra R, Atkinson PW et al. (2004) Transposition of hAT elements links transposable elements and V(D)J recombination. Nature 432(7020): 995–1001.

Further Reading

Atkinson H and Chalmers R (2010) Delivering the goods: viral and non‐viral gene therapy systems and the inherent limits on cargo DNA and internal sequences. Genetica 138(5): 485–498.

Craig NL (1997) Target site selection in transposition. Annual Review of Biochemistry 66: 437–474.

Craig NL, Craigie R, Gellert M and Lambowitz AM (2002) Mobile DNA II. Washington, DC: ASM Press.

Girard A and Hannon GJ (2008) Conserved themes in small‐RNA‐mediated transposon control. Trends in Cell Biology 18(3): 136–148.

Kleckner N (1990) Regulation of transposition in bacteria. Annual Review of Cell Biology 6: 297–327.

Le Provost F, Lillico S, Passet B et al. (2010) Zinc finger nuclease technology heralds a new era in mammalian transgenesis. Trends in Biotechnology 28(3): 134–141.

de Lencastre H, Oliveira D and Tomasz A (2007) Antibiotic resistant Staphylococcus aureus: a paradigm of adaptive power. Current Opinion in Microbiology 10(5): 428–435.

Nowotny M and Yang W (2009) Structural and functional modules in RNA interference. Current Opinion in Structural Biology 19(3): 286–293.

Palazzoli F, Testu FX, Merly F and Bigot Y (2010) Transposon tools: worldwide landscape of intellectual property and technological developments. Genetica 138(3): 285–299.

Rice PA and Correll CC (2008) Protein–Nucleic Acid Interactions: Structural Biology. Cambridge: Royal Society of Chemistry.

Contact Editor close
Submit a note to the editor about this article by filling in the form below.

* Required Field

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