Intermolecular and intramolecular transposition and transposition immunity in Tn3 and Tn2660

Summary Intermolecular transposition of Tn2660 into pCR1 was measured at 30°C in recA ^– and recA ⁺ hosts as between 2.6 and 5.5x10^–3, a similar value to that previously found for Tn3. No cointegrate structures were found under conditions where 10⁴ transposition events occurred. Immunity to intermolecular transposition of Tn2660, similar to that found for Tn3 was demonstrated by showing that the above transposition frequency was reduced by a factor of between 10^–3 and 10^–4 when a mutant Tn2660 (resulting in the synthesis of a temperaturesensitive -lactamase) was present in the recipient plasmid. Intramolecular transposition of Tn3 was found to occur under the same conditions as previously demonstrated for Tn2660 giving rise to similar end products, in which the newly introduced Tn3 is oriented inversely to the resident Tn3 and the DNA sequence between the two transposons has been inverted. Thus, in all respects functional identity of the transposition activities of Tn3 and Tn2660 is shown, thereby identifying characteristics of intramolecular transposition that are not readily accommodated by current models of transposition.     

Target DNA structure plays a critical role in Tn7 transposition

The bacterial transposon Tn7 utilizes four Tn7-encoded proteins, TnsA, TnsB, TnsC and TnsD, to make insertions at a specific site termed attTn7. This target is selected by the binding of TnsD to attTn7 in a sequence-specific manner, followed by the binding of TnsC and activation of the transposase. We show that TnsD binding to attTn7 induces a distortion at the 5' end of the binding site and TnsC contacts the region of attTn7 distorted by TnsD. Previous work has shown that a target site containing triplex DNA, instead of TnsD-attTn7, can recruit TnsABC and effect site- specific insertion of Tn7. We propose that the DNA distortion imposed by TnsD on attTn7, like the altered DNA structure via triplex formation, serves as a signal to recruit TnsC. We also show that TnsD primarily contacts the major groove of DNA, whereas TnsC is a minor groove binding protein. The footprint of the TnsC-TnsD-attTn7 nucleoprotein complex includes and extends beyond the Tn7 insertion site, where TnsC forms a platform to receive and activate the transposase to carry out recombination.     

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.     

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.     

DNA sequence bias during Tn5 transposition

Transposition is one of the primary mechanisms causing genome instability. This phenomenon is mechanistically related to other DNA rearrangements such as V(D)J recombination and retroviral DNA integration. In the Tn5 system, only one protein, the transposase (Tnp), is required for all of the catalytic steps involved in transposon movement. The complexity involved in moving multiple DNA strands within one active site suggests that, in addition to the specific contacts maintained between Tnp and its recognition sequence, Tnp also interacts with the flanking DNA sequence. Here, we demonstrate that Tnp interacts with the donor DNA region. Tnp protects the donor DNA from DNase I digestion, suggesting that Tnp is in contact with, or otherwise distorts, the donor DNA during synapsis. In addition, changes in the donor DNA sequence within this region alter the affinity of Tnp for DNA by eightfold during synapsis. In vitro selection for more stable synaptic complexes reveals an A/T sequence bias for this region. We further show that certain donor DNA sequences, which favor synapsis, also appear to serve as hot spots for strand transfer. The TTATA donor sequence represents the best site. Most surprising is the fact that this sequence is found within the Tnp recognition sequence. Preference for insertion into a site within the Tnp recognition sequence would effectively inactivate one copy of the element and form clusters of the Tn5 transposon. In addition, the fact that several donor DNA sequences, which favor synapsis, appear to serve as hot spots for transposon insertion suggest that similar criteria may exist for Tnp-donor DNA and Tnp-target DNA interactions.     

Mutation of Tn5 transposase beta-loop residues affects all steps of Tn5 transposition: the role of conformational changes in Tn5 transposition

X-ray cocrystal structures of Tn5 transposase (Tnp) bound to its 19 base pair (bp) recognition end sequence (ES) reveal contacts between a beta-loop (amino acids 240-260) and positions 3, 4, 5, and 6 of the ES. Here, we show that mutations of residues in this loop affect both in vivo and in vitro transposition. Most mutations are detrimental, whereas some mutations at position 242 cause hyperactivity. More specifically, mutations to the beta-loop affect every individual step of transposition tested. Mutants performing in vivo and in vitro transposition less efficiently also form fewer synaptic complexes, whereas hyperactive Tnps form more synaptic complexes. Surprisingly, two hypoactive mutations, K244R and R253L, also affect the cleavage steps of transposition with a much more dramatic effect on the second double end break (DEB) complex formation step, indicating that the beta-loop likely plays an important roll in positioning the substrate DNA within the catalytic site. Finally, all mutants tested decrease efficiency of the final transposition step, strand transfer. A disparity in cleavage rate constants in vitro for mutants with changes to the proline at position 242 on transposons flanked by ESs differing in the orientation of the A-T base pair at position 4 allows us to postulate that P242 contacts the position 4 nucleotide pair. On the basis of these data, we propose a sequential model for end cleavage in Tn5 transposition in which the uncleaved PEC is not symmetrical, and conformational changes are necessary between the first and second cleavage events and also for the final strand transfer step of transposition.     

In vitro transposition of transposon Tn3

Transposition of the ampicillin-resistant transposon Tn3 was reproduced in vitro using the Escherichia coli cell extract. In this cell-free system, we used plasmid DNA carrying mini-Tn3 as donor and phage lambda DNA as target and assayed for ampicillin-resistance transducing phages formed by cointegration of these DNA molecules. Ampicillin-resistance transducing phages, which were obtained by in vitro packaging of lambda DNA after the in vitro transposition reaction, were formed only in the presence of Tn3 transposase. The reaction required mini-Tn3 with the proper sequence and orientation of the terminal inverted repeats of Tn3. The reaction also required DNA synthesis but not RNA synthesis by E. coli RNA polymerase.     

Formation of a nucleoprotein complex containing Tn7 and its target DNA regulates transposition initiation

Tn7 insertion into its specific target site, attTn7, is mediated by the proteins TnsA, TnsB, TnsC and TnsD. The double-strand breaks that separate Tn7 from the donor DNA require the Tns proteins, the transposon and an attTn7 target DNA, suggesting that a prerequisite for transposition is the formation of a nucleoprotein complex containing TnsABC+D, and these DNAs. Here, we identify a TnsABC+D transposon-attTn7 complex, and demonstrate that it is a transposition intermediate. We demonstrate that an interaction between TnsB, the transposase subunit that binds to the transposon ends, and TnsC, the target DNA-binding protein that controls the activity of the transposase, is essential for assembly of the TnsABC+D transposon-attTn7 complex. We also show that certain TnsB residues are required for recombination because they mediate a TnsB-TnsC interaction critical to formation of the TnsABC+D transposon-attTn7 complex. We demonstrate that TnsA, the other transposase subunit, which also interacts with TnsC, greatly stabilizes the TnsABC+D transposon-attTn7 complex. Thus multiple interactions between the transposase subunits, TnsA and TnsB, and the target-binding transposase activator, TnsC, control Tn7 transposition.     

Tn5 as a model for understanding DNA transposition

Tn5 is an excellent model system for understanding the molecular basis of DNA-mediated transposition. Mechanistic information has come from genetic and biochemical investigations of the transposase and its interactions with the recognition DNA sequences at the ends of the transposon. More recently, molecular structure analyses of catalytically active transposase; transposon DNA complexes have provided us with unprecedented insights into this transposition system. Transposase initiates transposition by forming a dimeric transposase, transposon DNA complex. In the context of this complex, the transposase then catalyses four phosphoryl transfer reactions (DNA nicking, DNA hairpin formation, hairpin resolution and strand transfer into target DNA) resulting in the integration of the transposon into its new DNA site. The studies that elucidated these steps also provided important insights into the integration of retroviral genomes into host DNA and the immune system V(D)J joining process. This review will describe the structures and steps involved in Tn5 transposition and point out a biologically important although surprising characteristic of the wild-type Tn5 transposase. Transposase is a very inactive protein. An inactive transposase protein ensures the survival of the host and thus the survival of Tn5.     

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.     

Purification and characterization of TnsC, a Tn7 transposition protein that binds ATP and DNA.

The bacterial transposon Tn7 encodes five transposition genes tnsABCDE. We report a simple and rapid procedure for the purification of TnsC protein. We show that purified TnsC is active in and required for Tn7 transposition in a cell-free recombination system. This finding demonstrates that TnsC participates directly in Tn7 transposition and explains the requirement for tnsC function in Tn7 transposition. We have found that TnsC binds adenine nucleotides and is thus a likely site of action of the essential ATP cofactor in Tn7 transposition. We also report that TnsC binds non-specifically to DNA in the presence of ATP or the generally non-hydrolyzable analogues AMP-PNP and ATP-gamma-S, and that TnsC displays little affinity for DNA in the presence of ADP. We speculate that TnsC plays a central role in the selection of target DNA during Tn7 transposition.     

Fis plays a role in Tn5 and IS50 transposition.

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.     

Complementation of transposition of tnpA mutants of Tn3, Tn21, Tn501, and Tn1721

The transposons Tn21, Tn501, and Tn1721 are related to Tn3. Transposition-deficient mutants (tnpA) of these elements were used to test for complementation of transpostion. Transposition of tnpA mutants of Tn501 and Tn1721 was restored by the presence in trans of Tn21, Tn501, and Tn1721, but transposition of a tnpA mutant of Tn21 was restored in trans only by Tn21 itself. Tn3 did not complement transposition of Tn21, Tn501, or Tn1721, and these elements did not complement transposition of Tn3.     

Analysis of Tn3 sequences required for transposition and immunity

Tn3 is a 5-kb transposon (Tn) with 38-bp inverted terminal repeats (ITR). The two 38-bp terminal sequences are required in cis for Tn3 transposition. In this study, the role of the ITR in Tn3 transposition has been further dissected by the use of various mini-Tn3 Tn's. The transposition frequency of these mini-Tn's demonstrate that Tn3 contains no sequence other than the ITR sequences that are necessary for the first step in transposition; the two terminal repeats must be oriented as ITR for transposition to occur; the outside 34 bp of the ITR are required for transposition; and reducing the distance between the terminal sequences does not affect transposition frequency. Moreover, mutant copies of the ITR sequences that cannot function in transposition do not confer transposition immunity.     

Purification and characterisation of the TnsB protein of Tn7: a transposition protein that binds to the ends of Tn7.

Tn7, a large bacterial transposon encodes 5 proteins required for its transposition. We report a rapid and easy purification of one of these proteins, TnsB, from an overexpression strain. This protein was shown to bind to the ends of Tn7, in a bandshift assay, in two distinct stages as a function of protein concentration. DNasel footprinting at each end of Tn7 showed that the TnsB recognition sequence, a set of 22 bp repeats, plus Tn7 termini are protected. Binding of TnsB appeared cooperative but was only observed above a threshold concentration of protein. ATP and Mg2+ had no effect on the pattern of protection, nor did addition of other Tn7-encoded proteins. Hydroxyl radical footprinting, performed at the right end, showed that TnsB binds preferentially to one side of the DNA helix.     

Nucleotide sequences required for Tn3 transposition immunity.

The Tn3 transposon inserts at a reduced frequency into a plasmid already containing a copy of Tn3, a phenomenon known as transposition immunity. The cis-acting site on Tn3 responsible for immunity was mapped by deletions from each side to be within the terminal 38-base-pair sequence that is inversely repeated at the ends of Tn3. Two palindromic sequences are present in the essential part of this region. Some deletions conferred only partial immunity, and others conferred negative immunity. Multiple copies of partially immune ends conferred additional immunity. No other part of Tn3 was necessary for immunity.     

Kinetics of Tn5 transposition

The kinetics of Tn5 transposition and gene expression were studied. For about 2 h after infection with lambda Tn5, Tn5 transpositions accumulate, reaching a level of about 1.5% of the infected cells. After 2 h transposition is essentially turned off. In cells carrying a resident Tn5, transposition is undetectable after infection. The synthesis of the Tn5-specific proteins p58 and p54 and the kanamycin-resistance protein were studied in pre-irradiated cells infected with lambda Tn5. The synthesis of p58 and p54 peaked early after infection and was significantly reduced, relative to pneo, by 2 h after infection. Moreover, p54 appeared to reach a maximum later than p58. These kinetic data put new constraints on models for the regulation of Tn5 transposition.