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.     

Conformational isomerization in phage Mu transpososome assembly: effects of the transpositional enhancer and of MuB.

Initiation of phage Mu DNA transposition requires assembly of higher order protein-DNA complexes called Mu transpososomes containing the two Mu DNA ends and MuA transposase tetramer. Mu transpososome assembly is highly regulated and involves multiple DNA sites for transposase binding, including a transpositional enhancer called the internal activation sequence (IAS). In addition, a number of protein cofactors participate, including the target DNA activator MuB ATPase. We investigated the impact of the assembly cofactors on the kinetics of transpososome assembly with the aim of deciphering the reaction steps that are influenced by the cofactors. The transpositional enhancer IAS appears to have little impact on the initial pairing of the two Mu end segments bound by MuA. Instead, it accelerates the post-synaptic conformational step(s) that converts the reversible complex to the stable transpososome. The transpososome assembly stimulation by MuB does not require its stable DNA binding activity, which appears critical for directing transposition to sites distant from the donor transposon.     

MuB protein allosterically activates strand transfer by the transposase of phage Mu.

The MuA and MuB proteins collaborate to mediate efficient transposition of the phage Mu genome into many DNA target sites. MuA (the transposase) carries out all the DNA cleavage and joining steps. MuB stimulates strand transfer by activating the MuA-donor DNA complex through direct protein-protein contact. The C-terminal domain of MuA is required for this MuA-MuB interaction. Activation of strand transfer occurs irrespective of whether MuB is bound to target DNA. When high levels of MuA generate a pool of free MuB (not bound to DNA) or when chemical modification of MuB impairs its ability to bind DNA, MuB still stimulates strand transfer. However, under these conditions, intramolecular target sites are used exclusively because of their close proximity to the MuA-MuB-donor DNA complex.     

Differential role of the Mu B protein in phage Mu integration vs. replication: mechanistic insights into two transposition pathways

The Mu B protein is an ATP-dependent DNA-binding protein and an allosteric activator of the Mu transposase. As a result of these activities, Mu B is instrumental in efficient transposition and target-site choice. We analysed in vivo the role of Mu B in the two different recombination reactions performed by phage Mu: non-replicative transposition, the pathway used during integration, and replicative transposition, the pathway used during lytic growth. Utilizing a sensitive PCR-based assay for Mu transposition, we found that Mu B is not required for integration, but enhances the rate and extent of the process. Furthermore, three different mutant versions of Mu B, Mu BC99Y, Mu BK106A, and Mu B1-294, stimulate integration to a similar level as the wild-type protein. In contrast, these mutant proteins fail to support Mu growth. This deficiency is attributable to a defect in formation of an essential intermediate for replicative transposition. Biochemical analysis of the Mu B mutant proteins reveals common features: the mutants retain the ability to stimulate transposase, but are defective in DNA binding and target DNA delivery. These data indicate that activation of transposase by Mu B is sufficient for robust non-replicative transposition. Efficient replicative transposition, however, demands that the Mu B protein not only activate transposase, but also bind and deliver the target DNA.     

The dynamic Mu transpososome: MuB activation prevents disintegration

DNA transposases use a single active center to sequentially cleave the transposable element DNA and join this DNA to a target site. Recombination requires controlled conformational changes within the transposase to ensure that these chemically distinct steps occur at the right time and place, and that the reaction proceeds in the net forward direction. Mu transposition is catalyzed by a stable complex of MuA transposase bound to paired Mu DNA ends (a transpososome). We find that Mu transpososomes efficiently catalyze disintegration when recombination on one end of the Mu DNA is blocked. The MuB activator protein controls the integration versus disintegration equilibrium. When MuB is present, disintegration occurs slowly and transpososomes that have disintegrated catalyze subsequent rounds of recombination. In the absence of MuB, disintegration goes to completion. These results together with experiments mapping the MuA-MuB contacts during DNA joining suggest that MuB controls progression of recombination by specifically stabilizing a concerted transition to the “joining” configuration of MuA. Thus, we propose that MuB's interaction with the transpososome actively promotes coupled joining of both ends of the element DNA into the same target site and may provide a mechanism to antagonize formation of single-end transposition products.     

Gamma delta transposase and integration host factor bind cooperatively at both ends of gamma delta.

gamma delta, a prokaryotic transposon, encodes a transposase that is essential for its transposition. We show here, by DNase I protection experiments, that purified gamma delta transposase binds at the transposon's inverted repeats (IRs). Immediately adjacent to each transposase binding site (and within gamma delta DNA) we have identified a binding site for an additional protein factor, the Escherichia coli-encoded integration host factor (IHF). The binding of transposase and IHF to these adjacent sites is mutually cooperative. An IHF binding-site was also found in the original target DNA, just outside one of the ends of gamma delta. The affinity of IHF for this flanking site is reduced by transposase. These results demonstrate that gamma delta transposase binds at the IRs of gamma delta, and suggest that IHF may be involved in forming a transposase-DNA complex and/or influencing the target site selection during the transposition of gamma delta.     

Congruence of in vivo and in vitro insertion patterns in hot E. coli gene targets of transposable element Mu: opposing roles of MuB in target capture and integration

Phage Mu transposes promiscuously, employing protein MuB for target capture. MuB forms stable filaments on A/T-rich DNA, and a correlation between preferred MuB binding and Mu integration has been observed. We have investigated the relationship between MuB-binding and Mu insertion into hot and cold Mu targets within the Escherichia coli genome. Although higher binding of MuB to select hot versus cold genes was seen in vivo, the hot genes had an average A/T content and were less preferred targets in vitro, whereas cold genes had higher A/T values and were more efficient targets in vitro. These data suggest that A/T-rich regions are unavailable for MuB binding, and that A/T content is not a good predictor of Mu behavior in vivo. Insertion patterns within two hot genes in vivo could be superimposed on those obtained in vitro in reactions employing purified MuA transposase and MuB, ruling out the contribution of a special DNA structure or additional host factors to the hot behavior of these genes. While A/T-rich DNA is a preferred target in vitro, a fragment made up exclusively of A/T was an extremely poor target. A continuous MuB filament assembled along the A/T region likely protects it against the action of MuA. Our results suggest that MuB binds E. coli DNA in an interspersed manner utilizing local A/T richness, and facilitates capture of these bound regions by the transpososome. Actual integration events are then directed to sites that are in proximity to MuB filaments but are themselves free of MuB.     

Direct observation of single MuB polymers: evidence for a DNA-dependent conformational change for generating an active target complex

MuB, an ATP-dependent DNA binding protein, is critical for selection of target sites on the host chromosome during Mu transposition. We have developed a system for observing the behavior of single MuB polymers bound to an immobilized molecule of DNA. We show that the individual polymers display a broad distribution of disassembly rates and exhibit regional variations in DNA binding. Additionally, ATP hydrolysis was obligatorily coupled to dissociation of MuB subunits from the DNA during polymer disassembly. We propose a model in which the formation of an active target complex is mediated by a conformational change within the MuB polymer that is influenced by the sequence of the DNA.     

Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences

Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop. Using single-molecule and bulk biochemical approaches, we found that Tn5 transposase assembles a stable nucleoprotein complex in the absence of Tn5 transposon end sequences. Surprisingly, this end sequence-independent complex has structural similarities to the synaptic complex. This complex is the node of a DNA loop; transposase dimerization and DNA specificity mutants affect its assembly; and it likely has the same number of proteins and DNA molecules as the synaptic complex. Furthermore, our results indicate that Tn5 transposase preferentially binds and loops a subset of non-Tn5 end sequences. Assembly of end sequence-independent nucleoprotein complexes likely plays a role in the in vivo downregulation of transposition and the cis-transposition bias of many bacterial transposases.     

Analysis of gain-of-function mutants of an ATP-dependent regulator of Tn7 transposition

The bacterial transposon Tn7 is distinguished by its unusual discrimination among targets, being particularly attracted to certain target DNA and actively avoiding other DNA. Tn7 transposition is mediated by the interaction of two alternative transposon-encoded target selection proteins, TnsD and TnsE, with a common core transposition machinery composed of the transposase (TnsAB) and an ATP-dependent DNA-binding protein TnsC. No transposition is observed with wild-type TnsABC. Here, we analyze the properties of two gain-of-function TnsC mutants that allow transposition in the absence of TnsD or TnsE. We find that these TnsC mutants have altered interactions with ATP and DNA that can account for their gain-of-function phenotype. We also show that TnsC is an ATPase and that it directly interacts with the TnsAB transposase. This work provides strong support to the view that TnsC and its ATP state are central to the control of Tn7 transposition.     

Domain III function of Mu transposase analysed by directed placement of subunits within the transpososome

Assembly of the functional tetrameric form of Mu transposase (MuA protein) at the two att ends of Mu depends on interaction of MuA with multiple att and enhancer sites on supercoiled DNA, and is stimulated by MuB protein. The N-terminal domain I of MuA harbours distinct regions for interaction with the att ends and enhancer; the C-terminal domain III contains separate regions essential for tetramer assembly and interaction with MuB protein (IIIα and IIIβ, respectively). Although the central domain II (the ‘DDE’ domain) of MuA harbours the known catalytic DDE residues, a 26 amino acid peptide within IIIα also has a non-specific DNA binding and nuclease activity which has been implicated in catalysis. One model proposes that active sites for Mu transposition are assembled by sharing structural/catalytic residues between domains II and III present on separate MuA monomers within the MuA tetramer. We have used substrates with altered att sites and mixtures of MuA proteins with either wild-type or altered att DNA binding specificities, to create tetrameric arrangements wherein specific MuA subunits are nonfunctional in II, IIIα or IIIβ domains. From the ability of these oriented tetramers to carry out DNA cleavage and strand transfer we conclude that domain IIIα or IIIβ function is not unique to a specific subunit within the tetramer, indicative of a structural rather than a catalytic function for domain III in Mu transposition.     

Isolation and characterization of Tn7 transposase gain-of-function mutants: a model for transposase activation

Tn7 transposition has been hypothesized to require a heteromeric transposase formed by two Tn7-encoded proteins, TnsA and TnsB, and accessory proteins that activate the transposase when they are associated with an appropriate target DNA. This study investigates the mechanism of Tn7 transposase activation by isolation and analysis of transposase gain-of-function mutants that are active in the absence of these accessory proteins. This work shows directly that TnsA and TnsB are essential and sufficient components of the Tn7 transposase and also provides insight into the signals that activate the transposase. We also describe a protein-protein interaction between TnsA and TnsC, a regulatory accessory protein, that is likely to be critical for transposase activation.     

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.     

Effect of mutations in the C-terminal domain of Mu B on DNA binding and interactions with Mu A transposase

Bacteriophage Mu transposition requires two phage-encoded proteins, the transposase, Mu A, and an accessory protein, Mu B. Mu B is an ATP-dependent DNA-binding protein that is required for target capture and target immunity and is an allosteric activator of transpososome function. The recent NMR structure of the C-terminal domain of Mu B (Mu B223-312) revealed that there is a patch of positively charged residues on the solvent-exposed surface. This patch may be responsible for the nonspecific DNA binding activity displayed by the purified Mu B223-312 peptide. We show that mutations of three lysine residues within this patch completely abolish nonspecific DNA binding of the C-terminal peptide (Mu B223- 312). To determine how this DNA binding activity affects transposition we mutated these lysine residues in the full-length protein. The full-length protein carrying all three mutations was deficient in both strand transfer and allosteric activation of transpososome function but retained ATPase activity. Peptide binding studies also revealed that this patch of basic residues within the C-terminal domain of Mu B is within a region of the protein that interacts directly with Mu A. Thus, we conclude that this protein segment contributes to both DNA binding and protein-protein contacts with the Mu transposase.     

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.     

The Drosophila P-element KP repressor protein dimerizes and interacts with multiple sites on P-element DNA.

Drosophila P elements are mobile DNA elements that encode an 87-kDa transposase enzyme and transpositional repressor proteins. One of these repressor proteins is the 207-amino-acid KP protein which is encoded by a naturally occurring P element with an internal deletion. To study the molecular mechanisms by which KP represses transposition, the protein was expressed, purified, and characterized. We show that the KP protein binds to multiple sites on the ends of P-element DNA, unlike the full-length transposase protein. These sites include the high-affinity transposase binding site, an 11-bp transpositional enhancer, and, at the highest concentrations tested, the terminal 31-hp inverted repeats. The DNA binding domain was localized to the N-terminal 98 amino acids and contains a CCHC sequence, a potential metal binding motif. We also demonstrate that the KP repressor protein can dimerize and contains two protein-protein interaction regions and that this dimerization is essential for high-affinity DNA binding.