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{{short description|Enzyme that catalyzes movement of transposable elements}}
A '''transposase''' is any of a class of [[enzyme]]s capable of binding to the end of a [[transposon]] and catalysing its movement to another part of a [[genome]], typically by a cut-and-paste mechanism or a replicative mechanism, in a process known as transposition. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the [[Tn3 Transposon|Tn3 transposon]].<ref>{{cite journal | vauthors = Heffron F, McCarthy BJ, Ohtsubo H, Ohtsubo E | title = DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3 | journal = Cell | volume = 18 | issue = 4 | pages = 1153–63 | date = December 1979 | pmid = 391406 | doi = 10.1016/0092-8674(79)90228-9 | s2cid = 17775137 | doi-access =  }}</ref> The existence of transposons was postulated in the late 1940s by [[Barbara McClintock]], who was studying the inheritance of [[maize]], but the actual molecular basis for transposition was described by later groups. McClintock discovered that some segments of [[chromosome]]s changed their position, jumping between different loci or from one chromosome to another. The repositioning of these transposons (which coded for color) allowed other genes for pigment to be expressed.<ref name="website1">{{cite web | title = Transposase | url = http://pdb101.rcsb.org/motm/84 | work = Molecule of the Month | publisher = Protein Data Bank | date = December 2006 | first = David | last = Goodsell | name-list-style = vanc }}</ref> Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause [[Antimicrobial resistance|antibiotic resistance]].<ref name="website1" /> Transposition is also important in creating genetic diversity within species and generating adaptability to changing living conditions.<ref name="MM">{{cite journal | vauthors = Reznikoff WS | title = Tn5 as a model for understanding DNA transposition | journal = Molecular Microbiology | volume = 47 | issue = 5 | pages = 1199–206 | date = March 2003 | pmid = 12603728 | doi = 10.1046/j.1365-2958.2003.03382.x | doi-access = free }}</ref>

Transposases are classified under [[Enzyme Commission number|EC number]] EC 2.7.7. Genes encoding transposases are widespread in the genomes of most organisms and are the most abundant genes known.<ref>Aziz, R.K., M. Breitbart and R.A. Edwards (2010). Transposases are the most abundant, most ubiquitous genes in nature. ''Nucleic Acids Research'' 38(13): 4207–4217.{{cite journal | vauthors = Aziz RK, Breitbart M, Edwards RA | title = Transposases are the most abundant, most ubiquitous genes in nature | journal = Nucleic Acids Research | volume = 38 | issue = 13 | pages = 4207–17 | date = July 2010 | pmid = 20215432 | pmc = 2910039 | doi = 10.1093/nar/gkq140 }}</ref> During the course of human evolution, as much as 40% of the human genome has moved around via methods such as transposition of transposons.<ref name="website1" />

==Transposase Tn5==
{{Infobox protein family
| Symbol = Dimer_Tnp_Tn5
| Name = Transposase Tn5 dimerisation domain
| image = PDB 1mur EBI.jpg
| width = 
| caption = tn5 transposase: 20mer outside end 2 mn complex
| Pfam = PF02281
| Pfam_clan =  
| InterPro = IPR003201
| SMART = 
| PROSITE = 
| MEROPS = 
| SCOP = 1b7e
| TCDB = 
| OPM family = 
| OPM protein = 
| CAZy = 
| CDD = 
}}
Transposase (Tnp) Tn5 is a member of the [[Ribonuclease|RNase]] superfamily of proteins which includes retroviral [[integrase]]s. Tn5 can be found in ''[[Shewanella]]'' and ''[[Escherichia]]'' bacteria.<ref name="Website2">{{cite web | url = http://www.ebi.ac.uk/interpro/potm/2006_12/Page1.htm | title = Transposase | work = InterPro | first = Jennifer | last = McDowall | name-list-style = vanc }}</ref> The transposon codes for antibiotic resistance to [[kanamycin]] and other aminoglycoside antibiotics.<ref name="MM" /><ref name="N1">{{cite journal | vauthors = Lovell S, Goryshin IY, Reznikoff WR, Rayment I | title = Two-metal active site binding of a Tn5 transposase synaptic complex | journal = Nature Structural Biology | volume = 9 | issue = 4 | pages = 278–81 | date = April 2002 | pmid = 11896402 | doi = 10.1038/nsb778 | s2cid = 9721663 }}</ref>

Tn5 and other transposases are notably inactive. Because DNA transposition events are inherently mutagenic, the low activity of transposases is necessary to reduce the risk of causing a fatal mutation in the host, and thus eliminating the [[transposable element]]. One of the reasons Tn5 is so unreactive is because the N- and C-termini are located in relatively close proximity to one another and tend to inhibit each other. This was elucidated by the characterization of several mutations which resulted in hyperactive forms of transposases. One such mutation, L372P, is a mutation of amino acid 372 in the Tn5 transposase. This amino acid is generally a leucine residue in the middle of an alpha helix. When this leucine is replaced with a proline residue the alpha helix is broken, introducing a conformational change to the C-terminal domain, separating it from the N-terminal domain enough to promote higher activity of the protein.<ref name="MM" /> The transposition of a transposon often needs only three pieces: the transposon, the transposase enzyme, and the target DNA for the insertion of the transposon.<ref name="MM" /> This is the case with Tn5, which uses a cut-and-paste mechanism for moving around transposons.<ref name="MM" />

Tn5 and most other transposases contain a DDE motif, which is the active site that catalyzes the movement of the transposon. Aspartate-97, aspartate-188, and glutamate-326 make up the active site, which is a triad of acidic residues.<ref name="BC">{{cite journal | vauthors = Peterson G, Reznikoff W | title = Tn5 transposase active site mutations suggest position of donor backbone DNA in synaptic complex | journal = The Journal of Biological Chemistry | volume = 278 | issue = 3 | pages = 1904–9 | date = January 2003 | pmid = 12424243 | doi = 10.1074/jbc.M208968200 | doi-access =  free}}</ref> The DDE motif is said to coordinate divalent metal ions, most often magnesium and manganese, which are important in the catalytic reaction.<ref name="BC" /> Because transposase is incredibly inactive, the DDE region is mutated so that the transposase becomes hyperactive and catalyzes the movement of the transposon.<ref name="BC" /> The glutamate is transformed into an aspartate and the two aspartates into glutamates.<ref name="BC" /> Through this mutation, the study of Tn5 becomes possible, but some steps in the catalytic process are lost as a result.<ref name="MM" />
[[Image:1muh.jpg|right|thumb]]

There are several steps which catalyze the movement of the transposon, including Tnp binding, synapsis (the creation of a synaptic complex), cleavage, target capture, and strand transfer. Transposase then binds to the DNA strand and creates a clamp over the transposon end of the DNA and inserts into the active site. Once the transposase binds to the transposon, it produces a synaptic complex in which two transposases are bound in a cis/trans relationship with the transposon.<ref name="MM" />

In cleavage, the magnesium ions activate oxygen from water molecules and expose them to nucleophilic attack.<ref name="N1" /> This allows the water molecules to nick the 3' strands on both ends and create a hairpin formation, which separates the transposon from the donor DNA.<ref name="MM" /> Next, the transposase moves the transposon to a suitable location. Not much is known about the target capture, although there is a sequence bias which has not yet been determined.<ref name="MM" /> After target capture, the transposase attacks the target DNA nine base pairs apart, resulting in the integration of the transposon into the target DNA.<ref name="MM" />

As mentioned before, due to the mutations of the DDE, some steps of the process are lost—for example, when this experiment is performed ''in vitro'', and SDS heat treatment denatures the transposase. However, it is still uncertain what happens to the transposase ''in vivo''.<ref name="MM" />

The study of transposase Tn5 is of general importance because of its similarities to [[HIV]]-1 and other retroviral diseases. By studying Tn5, much can also be discovered about other transposases and their activities.<ref name="MM" />

Tn5 is utilized in genome sequencing by using the Tn5 to append sequencing adaptors and fragment the DNA in a single enzymatic reaction in 2010,<ref name=":0">{{cite journal |last1=Adey |first1=Andrew |title=Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition |journal=Genome Biology |date=December 2010 |volume=11 |issue=12 |article-number=R119 |doi=10.1186/gb-2010-11-12-r119 |pmid=21143862 |pmc= 3046479 |doi-access=free }}</ref> reducing the time and input requirements over traditional [[next-generation sequencing]] library preparation. The Tn5-based strategy can simplify the library preparation protocol significantly and can even can be incorporated into the direct colony-PCR for large numbers of bacterial isolates with no obvious coverage bias.<ref name=":0" /> The main disadvantages are less control of fragmented size compared to enzymatic fragmentation and mechanical fragmentation, and a bias toward high G-C content.<ref name=":0" /> This means of library preparation is also used in the [[ATAC-seq]] technique.

==Sleeping Beauty transposase==
The Sleeping Beauty (SB) transposase is the recombinase that drives the [[Sleeping Beauty transposon system]].<ref>{{cite journal | last1 = Ivics | first1 = Z. | last2 = Hackett | first2 = P.B. | last3 = Plasterk | first3 = R.A. | last4 = Izsvak | first4 = Z. | year = 1997 | title = Molecular reconstruction of Sleeping Beauty: a Tc1-like transposon from fish and its transposition in human cells | journal = Cell | volume = 91 | issue = 4| pages = 501–510 | doi = 10.1016/s0092-8674(00)80436-5 | pmid=9390559| doi-access = free }}</ref>  SB transposase belongs to the DD[E/D] family of transposases, which in turn belong to a large superfamily of polynucleotidyl transferases that includes RNase H, RuvC Holliday resolvase, RAG proteins, and retroviral integrases.<ref>{{cite journal | vauthors = Craig NL | title = Unity in transposition reactions | journal = Science | volume = 270 | issue = 5234 | pages = 253–4 | date = October 1995 | pmid = 7569973 | doi = 10.1126/science.270.5234.253 | bibcode = 1995Sci...270..253C | s2cid = 29930180 }}</ref><ref>{{cite journal | vauthors = Nesmelova IV, Hackett PB | title = DDE transposases: Structural similarity and diversity | journal = Advanced Drug Delivery Reviews | volume = 62 | issue = 12 | pages = 1187–95 | date = September 2010 | pmid = 20615441 | doi = 10.1016/j.addr.2010.06.006 | pmc=2991504}}</ref> The SB system is used primarily in vertebrate animals for gene transfer,<ref>{{cite journal | vauthors = Ivics Z, Izsvák Z | title = A whole lotta jumpin' goin' on: new transposon tools for vertebrate functional genomics | journal = Trends in Genetics | volume = 21 | issue = 1 | pages = 8–11 | date = January 2005 | pmid = 15680506 | doi = 10.1016/j.tig.2004.11.008 }}</ref> including gene therapy,<ref>{{cite journal | vauthors = Izsvák Z, Hackett PB, Cooper LJ, Ivics Z | title = Translating Sleeping Beauty transposition into cellular therapies: victories and challenges | journal = BioEssays | volume = 32 | issue = 9 | pages = 756–67 | date = September 2010 | pmid = 20652893 | pmc = 3971908 | doi = 10.1002/bies.201000027 }}</ref><ref>Aronovich, E.L., McIvor, R.S., and Hackett, P.B. (2011). The Sleeping Beauty transposon system – A non-viral vector for gene therapy. Hum. Mol. Genet. (in press){{cite journal | vauthors = Aronovich EL, McIvor RS, Hackett PB | title = The Sleeping Beauty transposon system: a non-viral vector for gene therapy | journal = Human Molecular Genetics | volume = 20 | issue = R1 | pages = R14-20 | date = April 2011 | pmid = 21459777 | pmc = 3095056 | doi = 10.1093/hmg/ddr140 }}</ref> and gene discovery.<ref>{{cite journal | vauthors = Carlson CM, Largaespada DA | title = Insertional mutagenesis in mice: new perspectives and tools | journal = Nature Reviews Genetics | volume = 6 | issue = 7 | pages = 568–80 | date = July 2005 | pmid = 15995698 | doi = 10.1038/nrg1638 | s2cid = 3194633 }}</ref><ref>{{cite journal | vauthors = Copeland NG, Jenkins NA | title = Harnessing transposons for cancer gene discovery | journal = Nature Reviews. Cancer | volume = 10 | issue = 10 | pages = 696–706 | date = October 2010 | pmid = 20844553 | doi = 10.1038/nrc2916 | s2cid = 6910577 }}</ref>  The engineered SB100X is an enzyme that directs the high levels of transposon integration.<ref>{{cite journal | vauthors = Mátés L, Chuah MK, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, Grzela DP, Schmitt A, Becker K, Matrai J, Ma L, Samara-Kuko E, Gysemans C, Pryputniewicz D, Miskey C, Fletcher B, VandenDriessche T, Ivics Z, Izsvák Z | display-authors = 6  | title = Molecular evolution of a novel hyperactive Sleeping Beauty transposase enables robust stable gene transfer in vertebrates | journal = Nature Genetics | volume = 41 | issue = 6 | pages = 753–61 | date = June 2009 | pmid = 19412179 | doi = 10.1038/ng.343 | s2cid = 27373372 }}</ref><ref>{{cite journal | vauthors = Grabundzija I, Irgang M, Mátés L, Belay E, Matrai J, Gogol-Döring A, Kawakami K, Chen W, Ruiz P, Chuah MK, VandenDriessche T, Izsvák Z, Ivics Z | title = Comparative analysis of transposable element vector systems in human cells | journal = Molecular Therapy | volume = 18 | issue = 6 | pages = 1200–9 | date = June 2010 | pmid = 20372108 | doi = 10.1038/mt.2010.47 | pmc=2889740}}</ref>

==Tn7 transposon==

The Tn7 transposon is a [[mobile genetic element]] found in many prokaryotes such as ''[[Escherichia coli]]'' (''E. coli''), and was first discovered as a DNA sequence in bacterial chromosomes and naturally occurring [[plasmids]] that encoded resistance to the antibiotics [[trimethoprim]] and [[streptomycin]].<ref>{{cite journal | vauthors = Barth PT, Datta N, Hedges RW, Grinter NJ | title = Transposition of a DNA sequence encoding trimethoprim and streptomycin resistances from R483 to other replicons.| journal = Journal of Bacteriology | volume = 125 | issue = 3 | pages = 800–810 | date = March 1976 | doi = 10.1128/JB.125.3.800-810.1976| pmid = 767328| pmc = 236152}}</ref><ref>{{cite journal | vauthors = Barth PT, Datta N | title = Two Naturally Occurring Transposons Indistinguishable from Tn7.| journal = Journal of General Microbiology | volume = 102 | issue = 1 | pages = 129–134 | date = Sep 1977 | pmid = 915473| doi = 10.1099/00221287-102-1-129| doi-access = free }}</ref> Specifically classified as a [[transposable element]] (transposon), the sequence can duplicate and move itself within a genome by utilizing a self-encoded recombinase enzyme called a transposase, resulting in effects such as creating or reversing mutations and changing genome size. The Tn7 transposon has developed two mechanisms to promote its propagation among prokaryotes.<ref name="Monika"/> Like many other bacterial transposons, Tn7 transposes at low-frequency and inserts into many different sites with little to no site-selectivity. Through this first pathway, Tn7 is preferentially directed into conjugable [[plasmids]], which can be replicated and distributed between bacteria. However, Tn7 is unique in that it also transposes at high-frequency into a single specific site in bacterial chromosomes called attTn7.<ref>{{cite journal | vauthors = Gringauz E, Orle KA, Waddell CS, Craig NL | title = Recognition of Escherichia coli attTn7 by transposon Tn7: lack of specific sequence requirements at the point of Tn7 insertion.| journal = Journal of Bacteriology | volume = 170 | issue = 6 | pages = 2832–2840 | date = Jun 1988 | pmid = 2836374| doi = 10.1128/jb.170.6.2832-2840.1988 | pmc = 211210}}</ref> This specific sequence is an essential and highly conserved gene found in many strains of bacteria. However, the recombination is not deleterious to the host bacterium as Tn7 actually transposes downstream of the gene after recognizing it, resulting in a safe way to propagate the transposon without killing the host. This highly evolved and sophisticated target-site selection pathway suggests this pathway evolved to promote coexistence between the transposon and it host, as well as Tn7's successful transmission into future generations of bacterium.<ref name ="Monika">{{cite journal | vauthors = Peters J, Craig NL | title = Tn7: smarter than we thought.| journal = Nature Reviews Molecular Cell Biology | volume = 2 | pages = 806–814 | date = Nov 2001 | issue = 11| pmid = 11715047| doi = 10.1038/35099006| s2cid = 34733892}}</ref>

The Tn7 transposon is 14 kb long and codes for five enzymes.<ref name ="Monika" /> The ends of the DNA sequence consists of two segments that the Tn7 transposase interacts with during recombination. The left segment (Tn7-L) is 150 bp long and the right sequence (Tn7-R) is 90 bp long. Both ends of the transposon contain a series of 22 bp binding sites that the Tn7 transposase recognizes and binds to. Within the transposon are five discrete genes encoding for proteins that make up the transposition machinery. In addition, the transposon contains an [[integron]], a DNA segment containing several [[gene cassette|cassettes]] of genes encoding for antibiotic-resistance.<ref name="Monika" />

The Tn7 transposon codes for five proteins: TnsA, TnsB, TnsC, TnsD, and TnsE.<ref name="Monika" /> TnsA and TnsB interact together to form the Tn7 transposase enzyme TnsAB. The enzyme specifically recognizes and binds to the ends of the DNA sequence of the transposon, and excises it by introducing double-stranded DNA breaks to each end. The excised sequence is then inserted to another target DNA site. Much like other characterized transposons, the mechanism for Tn7 transposition involves cleavage of the 3' ends from the donating DNA by the TnsA protein of the TnsAB transposase. However, Tn7 is also uniquely cleaved near the 5' ends, about 5 bp from the 5' end towards the Tn7 transposon, by the TnsB protein of TnsAB. After the insertion of the transposon into the target DNA site, the 3' ends are covalently linked to the target DNA, but the 5 bp gaps are still present at the 5' ends. As a result, repair of these gaps leads to a further 5 bp duplication at the target site. The TnsC protein interacts with the transposase enzyme and the target DNA to promote the excision and insertion processes. The ability of TnsC to activate the transposase depends on its interaction with a target DNA along with its appropriate targeting protein, TnsD or TnsE. The TnsD and TnsE proteins are alternative target selectors that are also DNA binding [[activator (genetics)|activators]] that promote excision and insertion of Tn7.  Their ability to interact with a particular target DNA is key to the target-site selection of Tn7. The proteins TnsA, TnsB, and TnsC thus form the core machinery of Tn7: TnsA and TnsB interact together to form the transposase, while TnsC functions as a [[regulator gene|regulator]] of the transposase's activity, communicating between the transposase and TnsD and TnsE.  When the TnsE protein interacts with the TnsABC core machinery, Tn7 preferentially directs insertions into conjugable plasmids. When the TnsD protein interacts with TnsABC, Tn7 preferentially directs insertions downstream into a single essential and highly conserved site in the bacterial chromosome. This site, attTn7, is specifically recognized by TnsD.<ref name="Monika" />

== References ==
{{reflist|33em}}

== External links ==
* {{MeshName|Transposases}}
* {{PDBe-KB2|Q46731|Transposase for transposon Tn5}}

{{Kinases}}
{{Enzymes}}
{{Portal bar|Biology|border=no}}

[[Category:EC 2.7.7]]
[[Category:Molecular biology]]
[[Category:Mobile genetic elements]]