Please note these web pages are part of an assignment for a graduate course in Advanced Biochemistry and Molecular Biology BCMB8010 at the University of Georgia. Questions should be directed to John Buchner

Background

Definitions and Common Features of Transposases

Transposition is defined by the relocation of specialized DNA elements, namely transposons and insertion elements, within or between chromosomes or extra chromosomal DNA mediated by either a transposase or an integrase, the enzymes of transposition. The substrate of transposition is DNA flanked by inverted repeats in its sequence that are recognized as the target for the transposase. The mobile stretch of DNA bordered by these inverted repeats is called the transposable element, the insertion sequence or the transposon. (3)

Transposases and integrases are enzymes that have conserved amino acids, tertiary structures, catalytic pathways, and metal ion requirements. For instance the aspartate, aspartate, glutamate (DDE) motif is found in most transposases and integrases (Table 1) and substitution of these amino acids eliminates catalytic activity. These residues are found to coordinate metal ions in the active sites. (3) Many transposases related to Tn5 transposase contain an element called the YREK (tyrosine, arginine, glutamate, lysine) signature. Alteration of the YREK residues reduces or eliminates transposition activity of these transposases. (1) A structural domain found in crystallized transposases and integrases is a mixed b sheet surrounded by a helices. The DDE and YREK motifs are found on these sheets, helices and loops of this domain. (1, 3) Other common features of transposasesπ tertiary structures are ions serving structural roles, multimerization domains, and DNA binding domains. (3)

		Location of Catalytic Amino Acid Residue in the Primary Sequence
Transposase/Integrase		Aspartate (D)	Aspartate (D)	Glutamate (E)
Tn5		97	188	326
HIV-1		64	116	152
Tn7		273	361	396
Mu		269	336	392
Table 1:	A comparison of transpoases, integrases, and the locations of the conserved DDE motif in the primary sequence. (3)

There are three basic steps of transposition: i) transposase binding to the inverted repeat sequences, ii) excision of the transposon from the host DNA, iii) integration of the transposon into the new locus of the DNA. The integration reaction creates small duplications of DNA at the site of integration. There are no covalently bound intermediates of transposition, and these steps are carried out isoenergeticaly, that is, without the consumption of nucleoside triphosphates. (3)

History and Relevance of Transposition

Transposons and insertion element have been found in a large cross section of organisms, but the first report of transposition came 50 years ago. In 1951, Barbara McClintock reported her observations of maize genetics at the Cold Spring Harbor Symposium in her ground breaking work entitled Chromosome Organization and Genetic Expression. This seminal work is the first description of transposition. McClintock was able to observe unusual behaviors of the chromatin of maize. She could see in micrographs of maize chromosomes the chromatin exchanging sections, resulting in the linkage of two chromosomes, and the uncoupling of other parts of the chromatin from the chromosome. This chromatin was aberrant in structure: some of the chromatin had two centromeres, or the centromere of some chromatin was absent, still other chromatin became ring shaped. Other chromatin abnormalities observed by McClintock were inversions, rearrangements, deletions and duplications. This aberrant chromatin did not always segregate properly in mitosis and meiosis. The chromatin without centromeres would be lost, and the double centromere chromosomes would be stretched in opposite directions until the chromatin that linked them snapped. McClintock desired to know the loci of these events, and named them Ds for ≥dissociation.≤ Her research uncovered something entirely unexpected. The loci for the alteration of chromatin moved. The movement of these loci she named ≥transposition.≤ They only transposed, however, in the presence of an Activator (Ac). McClintock observed two key features of Ac. First, the loci of Ac could not be mapped. Second although Ac was heritable, it did not observe Mendelian genetic laws. These two observations indicate Ac itself transposed. Another observation McClintock made was that each time one of these breaks occurred in chromosome 9, the phenotype of the maize kernels changed from the expected phenotype. Each change in kernel phenotype was the result of a new Ds transposition, but sometimes, the creation of a new phenotype by the insertion of Ds was reversible. McClintock observed that if transpositions happened at different stages of cell development, they could create across an ear, or even over a kernel, a wide variety of phenotypes. And finally, it was observed that some transpositions seemed to affect more than one phenotype (Figure 1). From this data McClintock deduced that: 1. The reversibility indicates the insertion of Ds into a gene created the alteration in phenotype, and the chromatin for the gene remained. 2. Some genes may be affecting the expression of other genes, or the alteration of genotype can affect not only the phenotype of that gene, but also the phenotype of the organism. 3. These transpositions can be a source of gene mutation, and play a role in an organismπs evolution. (2) All of these deductions have been borne out over the years, and today we know that the Ac locus is a transposable element encoding a transposase, and Ds is alternate locus of action for Ac. (14)

Human Immunodeficiency Virus Integrase-1 (HIV-1) shares many features with Tn5 transposase (1, 3, 15) (Figure 2). It has the DDE motif and a similar folding of the catalytic domain of the protein. (1, 3, 15) Positively charged metal ions are bound in the active site. (3) This integrase is able to form multimers. HIV-1 integrates the Human Immunodeficiency Virus DNA by binding the virus genome at the ends and catalyzing insertion into the host chromosome by nucleophilic attack isoenergeticaly. (3) It creates a small duplication upon integration. There is no covalently bound DNA protein intermediate in the reaction. (1, 3, 15)

Neisseria meningitidis causes sepsis and meningitis. One of the key virulence factors are the sialic acids of the lipooligosaccharide and the capsule. It has been discovered that an essential gene in the production of sialic acid, siaA can be reversibly regulated by the transposition of IS1301. (18) When this insertion element integrates, it creates a two base pair duplication. IS1301 excises in a manner homologous to Tn5 excision. (1, 3, 18) It cuts exactly at the interface of the insertion sequence and the host DNA. (3)

Tn5 was also called lbb kan-1, from its isolation in bacteriophage l, where it was determined to be a source of kanamycin resistance in bacteria. (19) It is a superb model system for transposition, and Tn5 transposase is an excellent model for a transposase.

The Substrate of Tn5 Transposase:

The substrate of Tn5 transposase is Tn5 (4) (Figure 3). In simplest terms, Tn5 is a length of DNA encoding antibiotic resistances flanked by two insertion sequences, IS50R and IS50L. Each IS50 is bordered by the inside end, the sequence adjoining the drug resistances, and the outside end, the sequence on the outside end of Tn5. Inside end and outside end sequences are similar, but not the same. They each are inverted repeats, and both can be recognized for binding by the transposase. (4) It has been demonstrated that interactions of the transposase and the outside end or inside end are sub optimal. (12) A hyperactive end, called the mosaic end, has been synthesized and used in some in vitro reactions. (20) Each end of IS50 is a substrate for host proteins. The outside end is recognized by DnaA, and the inside end is subject to dam methylation. IS50R encodes Tn5 transposase, and the polypeptide inhibitor of transposition (Inh). (4) The only substrate requirement for in vitro Tn5 transposition is a DNA of the proper length with the outside ends or the mosaic ends. (13, 20) The sequence of intervening DNA is unimportant, but DNA shorter than 64 base pairs cannot be transposed, and there is a periodic phenomena for DNA between 66 and 174 base pairs, with the period being 10.5 base pairs. This indicates that Tn5 transposase activity is related to transposase binding on the same face of the DNA. (11)

Tn5 Transposase Structure, Function and Purification:

The three dimensional architecture of Tn5 transposase has been solved by the crystallization of two proteins, an inhibitor of transposition (Inh) and a hyperactive mutant Tn5 transposase (Figure 4). (1,6)

The crystal structure of Inh, resolved to 2.9≈, was reported in 1999. Inh is synthesized from the same open reading frame as Tn5 transposase, but the start of translation is at the methionine codon at position 56. This results in a protein 421 amino acids long with the same primary amino acid sequence of Tn5 transposase, minus the first 55 amino acids. For simplicity, the numbering of amino acids in Inh corresponds to their position in Tn5 transposase; therefore, the first methionine in Inh is methionine 56. Inh can readily form homodimers in vitro, whereas Tn5 transposase does not. Inh cannot bind DNA in vitro, nor catalyze transposition. The heterodimerization of Inh with active Tn5 transposase inhibits transposition. This protein has been shown to be a trans dominant negative regulator of transposition. (6)

The co-crystal structure of hyperactive mutant of Tn5 transposase with the outside end of Tn5 bound in the active site, was recently resolved to 2.3≈. (1) This hyperactive mutant of Tn5 transposase has been crystallized because wild type transposase is inactive in vitro. (20) The mutations in this crystallized Tn5 transposase are glutamate 54 to lysine, methionine 56 to alanine, and leucine 327 to proline. (1) Interestingly, the glutamate and leucine mutations are shown to increase transposition frequency in vitro. (4) Naturally, the alteration of the methionine to alanine prevents the synthesis of Inh by eliminating the start codon of Inh. (1) The co-crystal Tn5 transposase also has one extra C-terminal glycine, an artifact of the purification system used. (1, 22)

For clarification purposes, the transposase will be described in three domains: the DNA binding N-terminus, the catalytic region, and the C-terminus the location of subunit interactions. All three domains must be functional for transposition to occur. (1)

The N-terminal domain:

The N-terminal domain (1, 17) (Figure 5a) of Tn5 transposase is required for Tn5 transposase binding, and transposition. If the N-terminal domain is non-functional or not present, transposition cannot occur. (1) Neither Inh nor a modified Tn5 transposase with the N-terminus cleaved (Tnpa) can bind DNA. (1, 6, 7) Tn5 transposase activity can be nullified or reduced by deletion of either the first eleven amino acids of Tn5 transposase, deletion of amino acids 30 through 35, or mutations of alanine 20, aspartate 24, arginine 30, or alanine 36 (1, 7) (Figure 5b).

The N-terminus of the hyperactive mutant transposase is comprised of four a helices (Figure 5a). Helices 3 and 4 are arranged in a helix turn helix motif; however, most the N-terminus residues that interact with the DNA are located on helix 4. Helix 4 is predicted to fit into the major groove between the seventh and twelfth base pairs on the DNA target. The arginine residues at positions 26, 27, and 30 (Figure 5b) on hyperactive mutant Tn5 transposase may form ionic interactions with the phosphates in the DNA backbone. The glutamate 54 to lysine (Figure 5b) mutation plays a role in binding as well. In wild type transposase, position 54 is close enough to interact with the oxygen in the fourth position on thymine 2. A negatively charged residue, like glutamate, would be repulsed by the negative charge on DNA, but a residue with protons to donate, such as lysine, would be attracted to DNA. The ability of the hyperactive mutant Tn5 transposase to have an increased rate of reaction in vitro is indicative that the poor DNA binding capability of wild type Tn5 transposase reduces the level of transposition in vivo. An important feature of catalysis of transposition is the N-terminus of Tn5 transposase binds the outside end opposite to the end to which it cuts. This is termed trans binding; because the DNA at the N-terminus binds is trans to the outside end the catalytic domain cuts (1) (Figure 6).

The Catalytic Domain

The catalytic domain is an a/b/a fold, with a mixed b sheet of five strands with strand 2 being anti-parallel to the other four strands (Figure 7a). It contains the trio of catalytic residues, aspartate 97, aspartate 188 and glutamate 326, the DDE motif. Substitution of any of these three key catalytic amino acids eliminates function. A Mn²⁺ in this co-crystal structure ion is bound in the active site, coordinated by aspartate 97 and glutamate 326, the 3πOH of the outside end of Tn5, and water (Figure 7b and c). The breaking of the phosphodiester bond in the DNA backbone at the 3π end is catalyzed by the presence of this Mn²⁺, and proceeds by nucleophilic attack. (1)

The catalytic site of the mutated Tn5 transposase, in addition to the DDE motif, also contains the YREK signature. (1) The YREK residues in Tn5 transposase are tyrosine 319, arginine 322, glutamate 326, and lysine 333 (6) (Figure 7b and c). Each residue of the YREK signature carries out an important function in transposition. Glutamate 326 (YREK) is one of the catalytic residues in the DDE motif. In the three dimensional structure, tyrosine 319 of YREK is in close proximity to the aspartate 188 of the catalytic residues. Substitution of the tyrosine 319 to phenylalanine results in an 83% reduction of activity. A tyrosine 319 to alanine substitution loses the ability to catalyze transposition. (6) The 5π phosphate in the DNA backbone in the active site is held stable by the amine group of arginine 210, the hydroxyl of tyrosine 319, and the Ne and N2 of arginine 322 of the YREK motif (1) (Figure 7c). Leading into the active site, there is a trench on the transposase for DNA binding. This trench has sixteen amino acids that make contact with the DNA. One of these amino acid residues is Lysine 333 (YREK). Lysine 333 and 330 interact with the same base in the DNA. Lysine 330 forms bonds with a cytosine moiety in the major groove, and lysine 333 interacts with the phosphate on the same strand. (1)

A DNA hairpin intermediate of transposition, created in the active site by the transposition reaction, is stabilized by tryptophan 298. The nucleoside of the thymine 2 is twisted out from its base pairing by formation of the transposition intermediate. Stacking with tryptophan 298 stabilizes this unusual backbone conformation (1) (Figure 7c).

A second set of transposase-DNA interactions occurs away from the catalytic site. The catalytic domain has cis contacts with the DNA substrate through arginine 342, arginine 344, and asparagine 348. These amino acid residues are part of a two strand anti-parallel b sheet (1) (Figure 7b). They are cis contact because these residues are interacting with the same end of insertion element DNA the catalytic domain cuts (Figure 6).

A second Mn²⁺ ion is bound to the hyperactive mutant Tn5 transposase between residues glutamate 110 and glutamate 345. This ion is on the end of a two helices, and confers stability upon the structure of the hyperactive mutant Tn5 transposase, evidenced by the ability to resolve crystal structure of the transposase at higher resolution when this ion is present (Figure 7b). A substitution of either the negatively charged glutamate 110 or glutamate 345 to a positively charged lysine increases the level of transposition, perhaps by creating a salt bridge between the wild type and mutant residue, and removes the Mn²⁺ requirement. (1)

It is interesting to note that although Inh has all the residues of the catalytic domain, the catalytic residues are not spaced correctly to catalyze the reaction, the b sheet that binds DNA is disordered in the crystal structure, and there is no apparent requirement for Mn²⁺ for the stability of the structure, demonstrated by the disordered diffraction of glutamate 345 in the crystal structure of Inh. (1, 6)

The C-terminus

A functional C-terminus (1, 17) (Figure 8) is not required for DNA binding, but a functional C-terminus is required for transposition. (1, 23) The C-terminus region of Tn5 is responsible for protein-protein interactions, but also binds DNA in trans. It is comprised entirely of a helices. (1) Inh has aided in resolving the residues involved in protein subunit interactions. One source of protein-protein interactions is found in the last helix of the protein. As a dimer, that helix, from serine 458 to methionine 470, sits at a 65 degree angle to its counterpart. These proteins come as close as 3.9≈ at glycine 462. In this helix, changing alanine 466 to aspartate eliminates the ability for Inh dimerization. (6) A deletion study of the C-terminus of Tn5 transposase found another region of interaction between the proteins. This study created two derivatives of Tn5 transposase, TnpD387 and TnpD369. TnpD387 had the last 89 C-terminal amino acids deleted. TnpD387 bound DNA, and bound other transposase monomers. TnpD369 has the last C-terminal 107 amino acids of Tn5 transposase deleted. TnpD369 is a protein that bound DNA, but could not bind other transposase monomers. This indicates another site of Tn5 transposase subunit interaction between residues of glutamate 387 and glutamate 396. (23) The substitution of leucine 372 to proline creates a break in a helix present in the wild type transposase. This disruption of geometry of the transposase increases the level of transposition because the C-terminus is suspected to interfere with the N-terminus DNA binding of wild type transposase. Because N-terminus binding is the first step of transposition, a wild type C-terminus interfering with N-terminus binding would lead to down regulation of transposition. (23) This mutation may also increase the dimerization potential of Tn5 transposase. (1)

Purification of Tn5 Transposase

The hyperactive mutant Tn5 transposase was purified using the IMPACT (Intein-Mediated Purification with an Affinity Chitin-binding Tag) system available from New England Biolabs. This system links the gene of interest to an intein-chitin binding domain tag. The chitin binding domain is an affinity tag with the ability to bind a column composed of chitin beads. Intein is a protein with the ability to recognize and cleave certain amino acid sequences under reducing conditions. (22) The IMPACT tag was fused to the C-terminus of the hyperactive mutant Tn5 transposase, under the control of an inducible T7 promoter on a plasmid and transformed into Esherichia coli ER2566. The cells were grown to mid-log phase, and then induced with 0.1mM isopropyl-1-thio-b-D-galactopyranoside. After a five hour room temperature incubation, the cells were harvested by centrifugation and suspended in TEGX (20mM Tris-HCl, pH 7.5, 0.7 M NaCl, 1mM EDTA, 10% Glycerol, 0.1% Triton X-100) and protease inhibitors. The cells were lysed by sonication. Unbroken cells and cellular debris were removed by centrifugation. The supernatant was loaded onto the chitin column. The column was washed with TEGX to remove non-specific binding. TEGX with 50mM dithiothreitol, a reducing agent, was used to wash the column and initiate intein cleavage. The column was incubated at 4 degrees C overnight. The Tn5 transposase was then eluted off the column with TEGX, and checked for purity. Tn5 transposase purified through this method was determined to be 95% pure by SDS-PAGE and Coomassie staining. (24) Note that although this method of purification is was used for the co-crystallization of Tn5 transposase with the outside end of Tn5, and was active for in vitro transposition, it is not necessarily the same method used to generate the Tn5 transposase in all of the in vitro reactions.

The Reaction:

Tn5 transposition can only occur through a homodimerization of active Tn5 transposase subunits bound to the outside end of Tn5. In order to suppress this, the IS50R element produces two inhibitors of transposition, Inh, and Tnpa. Inh can bind full length Tn5 transposase, and when it does, the Tn5 transposase will not be able to perform transposition. If Tn5 transposase is not bound to DNA in vivo, it becomes labile to proteolytic cleavage. Proteases process the Tn5 transposase, cleaving the approximately the first 25 amino acids off the N-terminus, eliminating DNA binding activity. The resulting protein is called Tnpa. When released from Inh, Tnpa can use its C-terminus to bind active monomers of Tn5 transposase in the same manner as Inh. It has been demonstrated that this action of Inh supports conversion of wild type Tn5 transposase to Tnpa. This is the first rule of wild type Tn5 transposition. The transposase must be supplied in cis to its substrate element. Any free wild type Tn5 transposase or naturally occurring variant of it will suppress transposition. (12)

Overproduction of wild type Tn5 transposase can be lethal to cells. This cell death is apparently involves the titration of the DNA binding protein topoisomerase I (Topo I), an enzyme used to relieve supercoiling of the DNA in bacterial cells. The presence of Tn5 transposase is correlated with more supercoiling of DNA in cells. An N-His₆-Tn5 transposase can be co-purified with Topo I, demonstrating a real interaction between those two proteins, whereas the same fusion on Inh does not co-purify with Topo I. This indicates that it is the N-terminal domain on Tn5 transposase that interacts with Topo I. (25)

Tn5 transposition occurs through what is called a cut and paste mechanism. There is no replication of this transposon required for transposition. The transposon in excised from the host, and integrated into the new locus as a simple insertion. Tn5 transposase is replicated by host cell machinery, but only when integrated into an inheritable element. The transposition reaction is divided into three parts, binding, excision, and integration. (1) Figure 9 illustrates the steps of transposition.

Binding

The first step in Tn5 transposition is the binding of the Tn5 transposase to the outside ends Tn5. (6) Methylation of the target DNA can inhibit binding, but the presence of the DnaA protein and active replication of DNA can promote transposition. (6, 12) The transposase contacts the outside ends through many amino acid residue/DNA base-pairing interactions, but the primary recognition of the outside ends occurs at the N-terminus. (1) As the transposase monomer binds DNA, it bends the DNA up to an angle of 116 degrees, and this bend in the DNA is at or near the point of catalysis. (4) There is evidence that there can be subunit exchange while the transposase is bound to DNA. (23) Western blot analysis of in vitro Tn5 transposase DNA binding assays has detected Inh and Tnpa dimerized to Tn5 transposase bound to DNA, when those proteins are present in the assay. (7) However, homodimers of Tn5 transposase are only detected if the solution is incubated over a long period of time, or with increasing concentrations of Tn5 transposase. This is indicative of how the protein inhibitors of transposition repress transposition not by interfering with DNA binding, but by not allowing homodimerization of bound Tn5 transposase. (6, 7, 26) In vitro binding assays display a sigmoidal curve for Tn5 transposase. The sigmoidal curve demonstrates that a subunit exchange may be occurring in transposition. (26)

Excision

Excision of the Tn5 is initiated by synaptic complex formation. Synaptic complex formation is when the two monomers bound to the outside end DNA are then brought into close proximity, where the C-termini interact. The DNA to be cleaved is fit into the catalytic site. This formation of the synaptic complex is highly favored. Each molecule of transposase is now bound to both ends of Tn5. The Tn5 transposase cleaves the outside end of the transposon opposite the outside end the N-terminus is bound to. This trans cutting is one way Tn5 can insure that both subunits are in place before cleavage. The cleavage reaction, catalyzed at each end by each subunit of the transposase, begins by taking a hydroxyl group from water, and using it to cleave the phosphodiester bond right at 3π end of the transposon. The free hydroxyl of the 3π end is then used to in nucleophilic attack the 5π strand phosphodiester bond. The cut is precisely at the border of the outside end of the transposon. No DNA from the host is taken with the transposon. Tn5 is free from that locus, and is in the hairpin intermediate. Presumably, another water is used to resolve the hairpin in the DNA, regenerating hydroxyl ends on the transposon DNA. (12) Figure 10 diagrams exicion

Integration

The first step of integration is target site selection. An in vitro transposition assay, where the only protein component is the hyperactive mutant Tn5 transposase, demonstrates that the only protein required for integration is the transposase. Analysis of locations of integrations have been examined in vivo with wild type transposase and in vitro and in vivo with the hyperactive mutant Tn5 transposase. Both proteins were determined to have similar site preferences. (9, 13)

In the current model, a molecule of Tn5 transposase not used in integration binds to the integration site. The transposase bound to the integration site has protein-protein interactions to guide the transposon-transposase complex into the integration site. No specific DNA sequence is required for Tn5 transposition, but site preference is exhibited by the transposase. The preferred sequence of Tn5 transposase for integration is A-GNT^T/_C^A/_T^A/_GANC-T where N is any base. One could imagine the site for integration as the combination of two half sites. That is, the Tn5 transposase recognizes a length of DNA as a target for integration, and the bases flanking the transposon after the insertion were part of that half site. Rather than seeing the integration site as one stretch of DNA, it is convenient to imagine the DNA as two halves to begin with. The most common half site for Tn5 transposase integration is GTTT, however, the complementary half site, AAAC, had a very low frequency of detection in vivo. The best complementary site in vivo combination is GGAT^A/_TATCC. GATC^A/_TGATC is the palindromic site most often detected in vivo. This last sequence is found in the promoter of Tn5 transposase and in the IS50 inside end (IE), a region Tn5 transposase can bind to, but integration has never been observed to occur in either of these locations. One feature of preferred targets of Tn5 transposase is having the sequence repeated many times over a length of DNA. To test these sequences plasmids were designed with the three pairs of sequences listed above in multiple adjacent copies. Each of the three plasmids was used in an in vitro transposition reaction, and the percentage of integration events occurring in the target sites was measured. For GTTT^A/_TAAAC^A/_T, 7% (2/29) of the integration events happened at the target site. The probability of that occurring by chance was 9.4x10^-3. 21.5% (14/65) of the integration events of the plasmid containing GGAT^A/_TATCC^A/_T occurred at the target site. The chance of finding that many inserts randomly was 1.8x10^-7. The plasmid containing the last target, GATC^A/_TGATC^A/_T was grown in strains that methylate DNA, and strains that do not. The methylation of the target sequence did not seem to affect the integration at all in this in vitro assay. 23% (21/92) of the integration events occurred at the target site where the expected random probability of this integration site was 5.5x10^-17. To summarize this data, Tn5 integration does not seem to be an entirely random process. The insensitivity of Tn5 transposase to methylation in the process of integration suggests a different DNA binding site on the transposase for integration, for it is known that methylation can inhibit Tn5 transposase binding for excision. Free Tn5 transposase may contain DNA binding sites not related to the DNA binding sites for transposition, and maintain protein interaction sites to guide the free transposon into the site of integration. (9)

Once a target is selected, the 3πOH ends of the transposon attacks the phosphodiester bonds of the target. In the case of Tn5 transposition, the attack of each 3πOH end is staggered by nine base pairs. Studies with Human Immunodeficiency Virus Integrase-1 indicate the phosphorus at the site of 3πOH attack goes through stereochemical inversion. The same may be happening with Tn5 transposition. This inversion of the stereochemistry of the phosphorus indicates the strand transfer process of transposition occurs in one step. (3) The product of this reaction is the transposon covalently bound, on its 3π ends to the integration site, flanked by nine base pairs of single stranded DNA of the integration site. This product can be used as a replication fork for host cell proteins to fill in the gaps created by integration. Because of the staggered nature of the insertion, this repair of the DNA creates nine base pair duplications of the DNA flanking the transposon. This duplication remains in the DNA after the transposon excises in the next cycle of transposition. (9) For Tn5 transposition, the integration reaction is so highly favored, the reversal of integration, disintegration, has never been detected for Tn5 transposase. (12) Figure 10 illustrates integration.

The removal of the transposase from Tn5 after integration is not well understood. In vitro, the hyperactive mutant Tn5 transposase stays bound to the product. Perhaps Tn5 transposase is similar to other transposases that depend on proteolysis for removal. (12)

The Application:

Tn5 transposition can be used to create deletions, inversion, and fusions of genes of interest. But, in order to understand how to go about this, an understanding of the in vitro Tn5 transposase is required.

The in vitro Tn5 assay (Figure 11) has three critical components: i) the transposase, ii) the DNA substrate, iii) the target DNA. The transposase chosen for in vitro assays is the hyperactive mutant Tn5 transposase because the hyperactive mutant Tn5 transposase was found to have 1000 times more activity than wild type transposase. pRZTL1, serving as both the target and substrate of transposition in this in vitro assay is a plasmid with kanamycin resistance and a synthetic transposon composed of: a bacterial origin of replication, chloramphenicol resistance, a silent tetracycline resistance, and Tn5 outside ends. The transposon for the assay is pRZTL1 minus the kanamycin resistance gene. The tetracycline resistance on the plasmid is silent until moved in front of a promoter. The design of the assay was to incubate hyperactive mutant Tn5 transposase and pRZTL1. The transposase excises the transposon from the plasmid. The next step is integration of the transposon, the remainder of pRZTL1, into the chloramphenicol resistance gene on a second copy of pRZTL1, with the goal of moving the tetracycline resistance gene into the proper location where it can be expressed. The integration must occur in the chloramphenicol resistance gene because the location of the promoter on pRZTL1 is in front of this gene. The products of the reaction are transformed into a E. coli strain, followed by selection for tetracycline resistance. This system worked to create many intermolecular, and the occasional intramolecular, products of transposition. Intramolecular products of in vitro transposition were observed when the DNA concentrations were low, 5ng DNA/ml, compared to the concentrations of DNA producing intermolecular products, 500ng DNA/ml (20).

The ability of hyperactive mutant Tn5 transposase is exploited for the creation of mutations and fusions (Figure 12). Once again, a plasmid is designed where most of the plasmid is the transposon. The transposon carries the gene of interest, the plasmid origin of replication, and ampicillin resistance, the mosaic ends, and stop codons adjacent to the outside end closest to the origin of replication. The fraction of the plasmid that is not a transposon is kanamycin resistance. The in vitro reaction opens with hyperactive mutant Tn5 transposase binding to the outside ends, and excising the transposon. The concentrations of plasmid in this assay are kept low, less than 2 fmol DNA/ul, so the target for integration becomes the transposon itself. The geometry of the attack determines if inversions or deletions will be created. The constraints of the reaction dictate only the outside end nearest to the origin of replication will be present on the proper intramolecular products. The products are then transformed in E. coli, and selected for ampicillin resistance and screened for kanamycin sensitivity. Proper intramolecular products will be kanamycin sensitive. This system could also be used to create tagged proteins by substitution of the stop codons with a molecular tag gene. (13)

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11. Goryshin, Igor Y., Kil, Yuri V., Reznikoff, William S., (1994) Proc. Natl. Acad. Sci. 91, 10834-10838 Pub Med

12. Reznikoff, William S., Bhasin, Archna, Davies, Douglas R., Goryshin, Igor Y., Mahnke, Lisa A. Nauman, Todd, Rayment Ivan, Steiniger-White, Mindy, Twinings, Sally S., (1999) Biochemical and Biophysical Research Communications 266, 729-734 Pub Med

13. York, Dona, Welch, Kelly, Goryshin, Igor Y., Reznikoff, William S., (1998) Nucleic Acids Research 26, 1927-1933 Pub Med

14. Fedoroff, Nina V., (1989) Maize Transposable Element p.380, in Mobile DNA, Berg and Howe, ASM, Washington DC

15. Molteni, Valentina, Greenwald, Jason, Rhodes, Denise, Hwang, Young, Kwiatkowski, Witek, Bushman, Fredric D., Siegel, Jay S., Choe, Senyon, (2001) Acta. Cryst. D57, 536-544 Pub Med

16. Sturcture 1HYV fro1m PDB

17. Sturcture 1F3I from PDB

18. Hammerschmidt, Sven, Hilse, Robert, van Putten, Jos, P.V., Gerardy-Schahn, Alexanfra Unkmeir, Frosch, Matthias, (1996) The EMBO Journal 15, 192-198 Pub Med

19. Berg, Douglas E., Davies, Julian, Aller, Bernard, Rochaix, Jeand-Davidson (1975) Proc. Natl. Acad. Sci. 72, 3628-3632

20. Goryshin, Igor Yu, Reznicoff, William S., (1998) The Journal of Biological Chemistry 273, No. 13 7367-7374Pub Med

21. Sturcture 1B7E from PDB

22. New England Biolabs IMPACT-CN version 1.7 (2001)

23. York, Donna, Reznikoff, William, S. (1997) Nucleic Acids Research, 1997, Vol. 25, No. 11 2153-2160 Pub Med

24. Bhasin, Archna, Goryshin, Igor, Y., Reznikoff, William S., (1999) The Journal of Biological Chemistry 274, No. 52, 37021-37029 Pub Med

25. Yigit, Hesna, Reznikoff, William S., (1999) Journal of Bacteriology, 181, No. 10, 3186-3192 Pub Med

26. York, Dona, Reznikoff, William S., (1996) Nucleic Acids Research, 24, No. 19 3790-3796 Pub Med

Please note these web pages are part of an assignment for a graduate course in Advanced Biochemistry and Molecular Biology BCMB8010 at the University of Georgia. Questions should be directed to John Buchner