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.     

Hairpin formation in Tn5 transposition

The initial chemical steps in Tn5 transposition result in blunt end cleavage of the transposon from the donor DNA. We demonstrate that this cleavage occurs via a hairpin intermediate. The first step is a 3' hydrolytic nick by transposase. The free 3'OH then attacks the phosphodiester bond on the opposite strand, forming a hairpin at the transposon end. In addition to forming precise hairpins, Tn5 transposase can form imprecise hairpins. This is the first example of imprecise hairpin formation on transposon end DNA. To undergo strand transfer, the hairpin must to be resolved by a transposase-catalyzed hydrolytic cleavage. We show that both precise and imprecise hairpins are opened by transposase. A transposition mechanism utilizing a hairpin intermediate allows a single transposase active site to cleave both 3' and 5' strands without massive protein/DNA rearrangements.     

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.     

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.     

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.     

Structure/function insights into Tn5 transposition

Prokaryotic transposon 5 (Tn5) serves as a model system for studying the molecular mechanism of DNA transposition. Elucidation of the X-ray co-crystal structure of Tn5 transposase complexed with a DNA recognition end sequence provided the first three-dimensional picture of an intermediate in a transposition/retroviral integration pathway. The many Tn5 transposase-DNA co-crystal structures now available complement biochemical and genetic studies, allowing a comprehensive and detailed understanding of transposition mechanisms. Specifically, the structures reveal two different types of protein-DNA contacts: cis contacts, required for initial DNA recognition, and trans contacts, required for catalysis. Protein-protein contacts required for synapsis are also seen. Finally, the two divalent metals in the active site of the transposase support a 'two-metal-ion' mechanism for Tn5 transposition.     

Tn5: A molecular window on transposition

DNA transposition is an underlying process involved in the remodeling of genomes in all types of organisms. We analyze the multiple steps in cut-and-paste transposition using the bacterial transposon Tn5 as a model. This system is particularly illuminating because of the existence of structural, genetic, and biochemical information regarding the two participating specific macromolecules: the transposase and the 19-bp sequences that define the ends of the transposon. However, most of the insights should be of general interest because of similarities to other transposition-like systems such as HIV-1 DNA integration into the host genome.     

Mutational analysis of the base flipping event found in Tn5 transposition.

This work identifies novel structure-function relationships between Tn5 transposase (Tnp) and its DNA recognition sequence. The Tn5 Tnp-DNA co-crystal structure revealed the protein-DNA contacts of the post-cleavage complex (Davies, D. R., Goryshin, I. Y., Reznikoff, W. S., and Rayment, I. (2000) Science 289, 77-85). One of the most striking features of this complex is the rotation of thymine 2 (T2) away from the DNA helix and into a pocket within the Tnp. This interaction appears similar to the "base flipping" phenomenon found in many DNA repair enzymes such as T4 endonuclease V and uracil DNA glycosylase (Roberts, R. J., and Cheng, X. (1998) Annu. Rev. Biochem. 67, 181-198). To study the biochemical significance of this phenomenon, we mutated the Tnp residues proposed to be involved in stabilizing this interaction and removed the T2 nucleotide to examine which steps in the transposition reaction require T2-Tnp interactions. From this work, we have determined that stacking interactions between T2 and Tnp are critical for efficient transposition in vitro. In addition, our results suggested that T2-Tnp interactions facilitate hairpin formation and hairpin resolution primarily through base stacking and that T2 plays a role in the alignment of the transposon DNA for strand transfer.     

Transposon Tn7. cis-Acting sequences in transposition and transposition immunity

We have identified and characterized the cis-acting sequences at the termini of the bacterial transposon Tn7 that are necessary for its transposition. Tn7 participates in two kinds of transposition event: high-frequency transposition to a specific target site (attTn7) and low-frequency transposition to apparently random target sites. Our analyses suggest that the same sequences at the Tn7 ends are required for both transposition events. These sequences differ in length and nucleotide structure: about 150 base-pairs at the left end (Tn7L) and about 70 base-pairs at the right end (Tn7R) are necessary for efficient transposition. We also show that the ends of Tn7 are functionally distinct: a miniTn7 element containing two Tn7R ends is active in transposition but an element containing two Tn7L ends is not. We also report that the presence of Tn7's cis-acting transposition sequences anywhere in a target replicon inhibits subsequent insertion of another copy of Tn7 into either an attTn7 target site or into random target sites. The inhibition to an attTn7 target site is most pronounced when the Tn7 ends are immediately adjacent to attTn7. We also show that the presence of Tn7R's cis-acting transposition sequences in a target replicon is necessary and sufficient to inhibit subsequent Tn7 insertion into the target replicon.     

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.     

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.     

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.     

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.     

Tn5-mediated transposition of plasmid DNA after transduction to Myxococcus xanthus.

After coliphage P1-mediated transfer of Tn5-containing plasmid DNA from Escherichia coli to Myxococcus xanthus, transductants were identified which contained plasmid sequences integrated at many sites on the bacterial chromosome. The unaltered plasmid DNA sequences in these transductants were apparently flanked by intact Tn5 or IS50 sequences. These results suggest that Tn5-mediated transposition has occurred and provide a method for integrating plasmid DNA into the M. xanthus chromosome without the requirement for homologous recombination.     

Integration host factor plays a role in IS50 and Tn5 transposition.

In Escherichia coli, the frequencies of IS50 and Tn5 transposition are greater in Dam- cells than in isogenic Dam+ cells. IS50 transposition is increased approximately 1,000-fold and Tn5 transposition frequencies are increased about 5- to 10-fold in the absence of Dam methylation. However, in cells that are deficient for both integration host factor (IHF) and Dam methylase, the transposition frequencies of IS50 and Tn5 approximate those found in wild-type cells. The absence of IHF alone has no effect on either IS50 or Tn5 transposition. These results suggest that IHF is required for the increased transposition frequencies of IS50 and Tn5 that are observed in Dam- cells. It is also shown that the level of expression of IS50-encoded proteins, P1 and P2, required for IS50 and Tn5 transposition and its regulation does not decrease in IHF- or in IHF- Dam- cells. This result suggests that the effects of IHF on IS50 and Tn5 transposition are not at the level of IS50 gene expression. Finally, IHF is demonstrated to significantly retard the electrophoretic mobility of a 289-base-pair segment of IS50 DNA that contains a putative IHF protein-binding site. The physiological role of this IHF binding site remains to be determined.     

Dissecting Tn5 transposition using HIV-1 integrase diketoacid inhibitors

Diketoacid (DKA) compounds have been shown to inhibit HIV-1 integrase by a mechanism that involves sequestration of the active site metals. Because HIV-1 integrase and Tn5 transposase have similar active site architectures and catalytic mechanisms, we investigated whether DKA analogues would inhibit Tn5 transposase activity and provide a model system to explore the mechanisms of action of these inhibitors. A screen of several hundred DKA analogues identified several with activity against Tn5 Tnp. Six DKA inhibitors used in this study manifested a variety of effects on different transposition steps suggesting that different analogues may have different binding contacts with transposase. All DKA compounds inhibited paired end complex (PEC) formation in which the nucleoprotein complex required for catalysis is assembled. Dissociation of PECs by some DKA compounds indicates that these inhibitors can decrease PEC stability. Four DKA compounds inhibited the two cleavage steps releasing transposon DNA from flanking DNA, and one of these four compounds preferentially inhibited the second cleavage step. The differential effect of this inhibitor on the second cleavage event indicates that cleavage of the two transposon-donor DNA boundaries is a sequential process requiring a conformational change. The requirement for a conformational change between cleavage events was also demonstrated by the inability of transposase to perform second cleavage at 25 degrees C. Finally, all six compounds inhibit strand transfer, the final step of Tn5 transposition. Two of the compounds that inhibited strand transfer have no effect on DNA cleavage. The strand transfer inhibition properties of various DKA compounds was sensitive to the structure of the 5'-non-transferred strand, suggesting that these compounds bind in or near the transposase active site. Other results that probe compound binding sites include the effects of active site mutations and donor DNA on DKA compound inhibition activities. Thus, DKA inhibitors will provide an important set of tools to investigate the mechanism of action of transposases and integrases.     

Intramolecular transposition by a synthetic IS50 (Tn5) derivative.

We report the formation of deletions and inversions by intramolecular transposition of Tn5-derived mobile elements. The synthetic transposons used contained the IS50 O and I end segments and the transposase gene, a contraselectable gene encoding sucrose sensitivity (sacB), antibiotic resistance genes, and a plasmid replication origin. Both deletions and inversions were associated with loss of a 300-bp segment that is designated the vector because it is outside of the transposon. Deletions were severalfold more frequent than inversions, perhaps reflecting constraints on DNA twisting or abortive transposition. Restriction and DNA sequence analyses showed that both types of rearrangements extended from one transposon end to many different sites in target DNA. In the case of inversions, transposition generated 9-bp direct repeats of target sequences.