Tn10 transposition and circle formation in vitro

We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event. Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires Tn10 transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. Tn10 is composed of inverted repeats of insertion sequence IS10. Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.     

Mutational analysis of the inverted repeats of Tn3

The transposase protein and the terminal inverted repeat sequences of the prokaryotic transposon Tn3 are essential for transposition. In order to determine the sequences within the inverted repeat necessary for transposition and interaction with transposase, we have constructed a series of mini-Tn3s in which specific mutations have been introduced into the inverted repeats. The effects of these mutations on transposition have been assayed in vivo using a mating-out transposition assay. Several single base-pair mutations within the transposase binding site reduce transposition frequency. Mutations that affect transposition show a greater effect when present in both inverted repeats than when present in only one inverted repeat.     

Mutations in the inverted repeats of Tn3 affect binding of transposase and transposition immunity.

In order to better understand the interaction between the inverted repeats (IRs) of the transposon Tn3 and Tn3 transposase, we have looked at the effects of mutations within the IRs on binding of transposase and transposition immunity. Binding of transposase to mutated IRs was measured using a site-specific nitrocellulose filter binding assay and by DNase I protection studies. Transposition immunity was measured in vivo using a transposition mating-out assay. The most important determinants for binding of transposase are present within the inside 21 base-pairs of the IR and several single base-pair mutations significantly reduce binding. Base-pair mutations which do not effect binding have strong negative effects on transposition immunity indicating that simple binding of transposase to the IR is not sufficient for the establishment of transposition immunity.     

Intramolecular transposition by Tn10

Transposon Tn10 promotes the formation of a circular product containing only transposon sequences. We show that these circles result from an intramolecular transposition reaction in which all of the strand cleavage and ligation events have occurred but newly created transposon/target junctions have not undergone repair. The unligated strand termini at these junctions are those expected according to a simple model in which the target DNA is cleaved by a pair of staggered nicks 9 bp apart, transposon sequences are separated from flanking donor DNA by cleavage at the terminal nucleotides on both strands (at both ends) of the element, and 3' transposon strand ends are ligated to 5' target strand ends. The stability of the unligated junctions suggests that they are protected from cellular processing by transposase and/or host proteins. We propose that the nonreplicative nature of Tn10 transposition is determined by the efficiency with which the nontransferred transposon strand is separated from flanking donor DNA and by the nature of the protein-DNA complexes present at the strand transfer junctions.     

Tn 10 transposition in vivo: temporal separation of cleavages at the two transposon ends and roles of terminal basepairs subsequent to interaction of ends.

During Tn10 transposition, the transposon is fully excised from the donor site by double strand cleavages at the two ends of the element prior to integration at a new target site. Results presented here demonstrate that an interaction between the two transposon ends is required for double strand cleavage at either end. Furthermore, despite this essential interaction of ends, subsequent cleavages at the two ends can occur at observably distinct times prior to occurrence of strand transfer at either end. Moreover, the time between cleavages at the two ends is exaggerated by the presence of an appropriate mutation at one end of the element. Biological rationales for this constellation of mechanistic features are suggested. Additional results demonstrate that mutations at the three terminal basepairs of Tn10 confer defects subsequent to interaction of ends, in confirmation of inferences from genetic analysis. More specifically, mutations in bp 1-3 confer strong defects during conversion of the full excision intermediate to a complete strand transfer product; mutations in bp 1 and 2 also confer more subtle defects subsequent to interaction of ends but prior to full excision. Such defects might reflect roles for these basepairs in the chemical steps of transposition per se, the positioning of terminal residues for those chemical steps, and/or the coupling of cleavage(s) to subsequent conformational changes.     

Tn3 transposition immunity is conferred by the transposase-binding domain in the terminal inverted-repeat sequence of Tn3

A series of mutant terminal inverted repeats (IRs), having 2 bp substitutions at various sites within the 38-bp IR sequence of the ampicillin-resistance transposon Tn3, were tested for transposition immunity to Tn3. Mutations within region 1-10 in the IR did not affect transposition immunity, while mutations within region 13-38 inactivated the immunity function. These two regions corresponded to domain A which was not bound specifically by Tn3 transposase and to domain B which was bound by the transposase, respectively. This indicates that specific binding of transposase to domain B within the IR sequence is responsible for transposition immunity.     

DNA-binding activity and subunit interaction of the mariner transposase

Mos1 is a member of the mariner/Tc1 family of transposable elements originally identified in Drosophila mauritiana. It has 28 bp terminal inverted repeats and like other elements of this type it transposes by a cut and paste mechanism, inserts at TA dinucleotides and codes for a transposase. This is the only protein required for transposition in vitro. We have investigated the DNA binding properties of Mos1 transposase and the role of transposase–transposase interactions in transposition. Purified transposase recognises the terminal inverted repeats of Mos1 due to a DNA-binding domain in the N-terminal 120 amino acids. This requires a putative helix–turn–helix motif between residues 88 and 108. Binding is preferentially to the right hand end, which differs at four positions from the repeat at the left end. Cleavage of Mos1 by transposase is also preferentially at the right hand end. Wild-type transposase monomers interact with each other in a yeast two-hybrid assay and we have used this to isolate mutations resulting in reduced interaction. These mutations lie along the length of the protein, indicating that transposase–transposase interactions are not due to a single interaction domain. One such mutation which retains both DNA-binding and catalytic activity has greatly reduced ability to excise Mos1 from plasmid DNA through coordinate cleavage of the two ends and transposition in vitro is lowered to a level 20-fold below that of the wild-type. This suggests that transposase–transposase interaction is required to form a synaptic complex necessary for coordinate cleavage at the ends of Mos1 during transposition. This mutant enzyme allows insertion at dinucleotides other than TA, including sequences with GC base pairs. This is the first example of a mariner/Tc1 transposase with altered target specificity.     

DNA sequence analysis of the transposon Tn3: three genes and three sites involved in transposition of Tn3.

The complete nucleotide sequence of the transposon Tn3 and of 20 mutations which affect its transposition are reported. The mutations, generated in vitro by random insertion of synthetic restriction sites, proved to contain small duplications or deletions immediately adjacent to the new restriction site. By determining the phenotype and DNA sequence of these mutations we were able to generate an overlapping phenotypic and nucleotide map. This 4957 bp transposon encodes three polypeptides which account for all but 350 bp of its total coding capacity. These proteins are the transposase, a high molecular weight polypeptide (1015 amino acids) encoded by the tnpA gene; the Tn3-specific repressor, a low molecular weight polypeptide (185 amino acids) encoded by the tnpR gene; and the 286 amino acid beta-lactamase. The 38 bp inverted repeats flanking Tn3 appear to be absolutely required in cis for Tn3 to transpose. Genetic data suggest that Tn3 contains a third site (Gill et al., 1978), designated IRS (internal resolution site), whose absence results in the insertion of two complete copies of Tn3 as direct repeats into the recipient DNA. We suggest that these direct repeats of complete copies of Tn3 are intermediates in transposition, and that the IRS site is required for recombination and subsequent segregation of the direct repeats to leave a single copy of Tn3 (Gill et al., 1978). A 23 nucleotide sequence within the amino terminus of the transposase which shares strong sequence homology with the inverted repeat may be the internal resolution site.     

Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism

The bacterial Tn5 and Tn10 transposases have a single active site that cuts both strands of DNA at their respective transposon ends. This is achieved using a hairpin intermediate that requires the DNA to change conformation during the reaction. In Tn5 these changes are controlled in part by a flipped nucleoside that is stacked on a tryptophan residue in a hydrophobic pocket of the transposase. Here we have investigated the base flipping mechanism in Tn10 transposition. As in Tn5 transposition, we find that base flipping takes place after the first nick and is required for efficient hairpin formation and resolution. Experiments with an abasic substrate show that the role of base flipping in hairpin formation is to remove the base from the DNA helix. Specific interactions between the flipped base and the stacking tryptophan residue are required for hairpin resolution later in the reaction. We show that base flipping in Tn10 transposition is not a passive reaction in which a spontaneously flipped base is captured and retained by the protein. Rather, it is driven in part by a methionine probe residue that helps to force the flipped base from the base stack. Overall, it appears that base flipping in Tn10 transposition is similar to that in Tn5 transposition.     

Identification and characterization of a pre-cleavage synaptic complex that is an early intermediate in Tn10 transposition.

The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.     

Factors responsible for target site selection in Tn10 transposition: a role for the DDE motif in target DNA capture.

Tn10, like several other transposons, exhibits a marked preference for integration into particular target sequences. Such sequences are referred to as integration hotspots and have been used to define a consensus target site in Tn10 transposition. We demonstrate that a Tn10 hotspot called HisG1, which was identified originally in vivo, also functions as an integration hotspot in vitro in a reaction where the HisG1 sequence is present on a short DNA oligomer. We use this in vitro system to define factors which are important for the capture of the HisG1 target site. We demonstrate that although divalent metal ions are not essential for HisG1 target capture, they greatly facilitate capture of a mutated HisG1 site. Analysis of catalytic transposase mutants further demonstrates that the DDE motif plays a critical role in ''divalent metal ion-dependent'' target capture. Analysis of two other classes of transposase mutants, Exc+ Int- (which carry out transposon excision but not integration) and ATS (altered target specificity), demonstrates that while a particular ATS transposase binds HisG1 mutants better than wild-type transposase, Exc+ Int- mutants are defective in HisG1 capture, further defining the properties of these classes of mutants. Possible mechanisms for the above observations are considered.     

Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains.

Two mutations within the transposase (the A protein) gene of phage Mu with distinct effects on DNA transposition have been studied. The first mutation maps to the central domain (domain II) of A, a protein consisting of three major structural domains. The variant protein is normal in synapsis and cleavage of Mu ends but is temperature-sensitive in the strand transfer reaction, joining the Mu ends to target DNA. The second mutation is a deletion at the C terminus (within domain III); on the basis of genetic studies, the mutant protein is predicted to have lost the ability to interact with the Mu B protein. The B protein, in conjunction with A, promotes efficient intermolecular transposition, while inhibiting intramolecular transposition. We show that the purified mutant protein is proficient in intramolecular, but not intermolecular transposition in vitro. The interactions between A and B proteins have been followed by a proteolysis assay. The chymotrypsin sensitivity of the interdomainal Phe221-Ser222 peptide bond within the bidomainally organized B protein is exquisitely modulated by ATP, DNA and A protein. The sensitive or "open" state of this bond in native B protein becomes partially "open" upon binding of ATP by B, attains a "closed" or resistant configuration upon binding of DNA in presence of ATP, and is rendered "open" again upon addition of the A protein. In this test for the interaction of A protein with B protein-DNA complex, the domain II mutant behaves like wild-type A protein. However, the domain III mutant fails to restore chymotrypsin susceptibility of the Phe221-Ser222 bond.     

The N-terminus of Himar1 mariner transposase mediates multiple activities during transposition

Mariner family transposons are perhaps the most widespread transposable elements of eukaryotes. While we are beginning to understand the precise mechanism of transposition of these elements, the structure of their transposases are still poorly understood. We undertook an extensive mutagenesis of the N-terminal third of the transposase of the Himar1 mariner transposon to begin the process of determining the structure and evolution of mariner transposases. N and C-terminal deletion analyses localized the DNA binding domain of Himar1 transposase to the first 115 amino acids. Alanine scanning of 23 selected sites within this region uncovered mutations that not only affected DNA binding but DNA cleavage as well. The behavior of other mutations strongly suggested that the N-terminus is also involved in multimerization of the transposase on a single inverted terminal repeat and in paired ends complex formation which brings together the two ends of the transposon. Finally, two hyperactive mutations at conserved sites suggest that mariner transposases are under a pattern of stabilizing selection in nature with regard to how efficiently they mediate transposition, resulting in a population of “average” transposons.     

Transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo

We present evidence that Tn10 transposase promotes double strand breaks and single strand joints at Tn10 termini in vivo. Plasmids containing a shortened Tn10 element and a transposase overproducer fusion give rise, upon transposase induction, to new DNA species. The most prominent class is a circularized transposon molecule whose structure suggests that it arises from double strand breakage at the two transposon ends followed by covalent joining between the 3' and 5' ends of one of the two strands. We have used formation of the circularized transposon as a physical assay for the interaction between transposase and different mutant and wild-type termini. These experiments show that transposase protein interacts preferentially with the genetically most active termini in a way that suppresses productive interaction with weaker termini present on the same substrate molecule.     

dnaA, an essential host gene, and Tn5 transposition.

Mutations in dnaA, an essential gene in Escherichia coli, decrease the frequency of transposition of Tn5. An insertion mutation in the dnaA gene does not affect Tn5 gene expression. Therefore, the DnaA protein plays a role either in the transposition reaction itself or in some type of cellular regulation of transposition. Analysis of a mutation in the DnaA box, found at the outside end of IS50, is consistent with a direct interaction of the protein through these bases. IS50 transposition, which utilizes only one end containing a DnaA box, is not affected by dnaA mutations. Overproduction of the DnaA protein does not increase transposition frequencies in wild-type cells, even when the transposase is also overproduced.     

Alternative interactions between the Tn7 transposase and the Tn7 target DNA binding protein regulate target immunity and transposition

The Tn7 transposon avoids inserting into a target DNA that contains a pre-existing copy of Tn7. This phenomenon, known as 'target immunity', is established when TnsB, a Tn7 transposase subunit, binds to Tn7 sequences in the target DNA and mediates displacement of TnsC, a critical transposase activator, from the DNA. Paradoxically, TnsB-TnsC interactions are also required to promote transposon insertion. We have probed Tn7 target immunity by isolating TnsB mutants that mediate more frequent insertions into a potentially immune target DNA because they fail to provoke dissociation of TnsC from the DNA. We show that a single region of TnsB mediates the TnsB-TnsC interaction that underlies both target immunity and transposition, but that TnsA, the other transposase subunit, channels the TnsB-TnsC interaction toward transposition.     

Role of the IS50 R proteins in the promotion and control of Tn5 transposition

IS50R, the inverted repeat sequence of Tn5 which is responsible for supplying functions that promote and control Tn5 transposition, encodes two polypeptides that differ at their N terminus. Frameshift, in-frame deletion, nonsense, and missense mutations within the N terminus of protein 1 (which is not present in protein 2) were isolated and characterized. The properties of these mutations demonstrate that protein 1 is absolutely required for Tn5 transposition. None of these mutations affected the inhibitory activity of IS50, confirming that protein 2 is sufficient to mediate inhibition of Tn5 transposition. The effects on transposition of increasing the amount of protein 2 (the inhibitor) relative to protein 1 (the transposase) were also analyzed. Relatively large amounts of protein 2 were required to see a significant decrease in the transposition frequency of an element. In addition, varying the co-ordinate synthesis of the IS50 R proteins over a 30-fold range had little effect on the transposition frequency. These studies suggest that neither the wild-type synthesis rate of protein 2 relative to protein 1 nor the amount of synthesis of both IS50 R proteins is the only factor responsible for controlling the transposition frequency of a wild-type Tn5 element in Escherichia coli.     

Determinants for hairpin formation in Tn10 transposition

Tn10 transposition involves the formation of a hairpin intermediate at the transposon termini. Here we show that hairpin formation exhibits more stringent DNA sequence requirements at the terminal two base pairs than either transpososome assembly or first strand nicking. We also observe a significant DNA distortion at the terminal base pairs upon transpososome assembly by chemical nuclease footprinting. Interestingly, mutations at these positions do not necessarily inhibit the formation of the distortion. However, it remains a possibility that the inhibitory effect of these mutations is due to a defect in protein-DNA interactions subsequent to this deformation. Terminal base pair mutations also inhibited strand transfer, providing evidence that transposase interactions with the terminal residues on both 'transferred' and 'non-transferred' strands are important for hairpin formation. We also demonstrate that mutation of a highly conserved tyrosine residue that is a component of the YREK motif, Y285, results in a phenotype comparable to that of the terminal base pair mutations. In contrast, a mutation at another conserved position, W265, is shown to relax the specificity of the hairpin formation reaction.