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.     

Criss-crossed interactions between the enhancer and the att sites of phage Mu during DNA transposition.

A bipartite enhancer sequence (composed of the O1 and O2 operator sites) is essential for assembly of the functional tetramer of phage Mu transposase (MuA) on supercoiled DNA substrates. A three-site interaction (LER) between the left (L) and right (R) ends of Mu (att sites) and the enhancer (E) precedes tetramer assembly. We have dissected the role of the enhancer in tetramer assembly by using two transposase proteins that have a common att site specificity, but are distinct in their enhancer specificity. The activity of these proteins on substrates containing hybrid enhancers reveals a 'criss-crossed' pattern of interaction between att and enhancer sites. The left operator, O1, of the enhancer interacts specifically with the transposase subunit at the R1 site (within the right att sequence) that is responsible for cleaving the left end of Mu. The right operator, O2, shows a preferential interaction with the transposase subunit at the L1 site (within the left att sequence) that is responsible for cleaving the right end of Mu.     

The same two monomers within a MuA tetramer provide the DDE domains for the strand cleavage and strand transfer steps of transposition.

The chemistry of Mu transposition is executed within a tetrameric form of the Mu transposase (MuA protein). A triad of DDE (Asp, Asp35Glu motif) residues in the central domain of MuA (DDE domain) is essential for both the strand cleavage and strand transfer steps of transposition. Previous studies had suggested that complete Mu transposition requires all four subunits in the MuA tetramer to carry an active DDE domain. Using a mixture of MuA proteins with either wild-type or altered att-DNA binding specificities, we have now designed specific arrangements of MuA subunits carrying the DDE domain. From analysis of the abilities of oriented tetramers to carry out DNA cleavage and strand transfer from supercoiled DNA, a new picture of the disposition of DNA and protein partners during transposition has emerged. For DNA cleavage, two subunits of MuA located at attL1 and attR1 (sites that undergo cleavage) provide DDE residues in trans. The same two subunits contribute DDE residues for strand transfer, also in trans. Thus, only two active DDE+ monomers within the tetramer carry out complete Mu transposition. We also show that when the attR1-R2 arrangement used on supercoiled substrates is tested for cleavage on linear substrates, alternative chemically competent DNA-protein associations are produced, wherein the functional DDE subunits are positioned at R2 rather than at R1.     

Mutational analysis of the att DNA-binding domain of phage Mu transposase.

The transposase (A protein) of phage Mu encodes binding to two families of DNA sites, att sites located at the Mu ends and enhancer sites located internally. Separate subdomains in the N-terminal domain I of Mu A protein are known to be involved in recognition of the att and enhancer sites. We have delineated an approximately 135 aa region within domain I beta gamma that specifies binding to Mu att sites. This peptide was overexpressed and its properties compared with that of the larger domain I beta gamma as well as the intact Mu A protein. Extensive mutagenesis of residues around a putative helix-turn-helix DNA-binding motif within the I beta domain identified several mutants defective in DNA transposition in vivo. Of these, Mu A(K157Q) was completely defective in att DNA-binding. Mu A(F131S) and Mu A(R146N) had a lower affinity for att DNA and low levels of transposition in vitro. Our results indicate that residues in the gamma region are required for activity and that residues outside the beta gamma region must also influence discrimination between the multiple att sites.     

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.     

DNA-protein complexes during attachment-site synapsis in Mu DNA transposition.

Initial events in Mu DNA transposition involve specific recognition of Mu DNA ends (att sites) and an internal enhancer site by the Mu transposase (A protein). This interaction between A protein and Mu DNA sequences present on a supercoiled DNA substrate leads to the formation of a stable synaptic complex in which the att ends are nicked, prior to DNA strand transfer. This study examines the properties of a synaptic complex proficient for DNA transposition. We show that the A protein binds as a monomer to its binding sites, and causes the DNA to bend through approximately 90 degrees at each site. All six att binding sites (three at each Mu end) are occupied by A within the synaptic complex. Three of these sites are loosely held and can be emptied of A upon challenge with heparin. A synaptic complex with only three sites occupied is stable and is fully competent in the subsequent strand-transfer step of transposition.     

DNA recognition sites activate MuA transposase to perform transposition of non-Mu DNA.

Mu transposition occurs within a large protein-DNA complex called a transpososome. This stable complex includes four subunits of MuA transposase, each contacting a 22-base pair recognition site located near an end of the transposon DNA. These MuA recognition sites are critical for assembling the transpososome. Here we report that when concentrations of Mu DNA are limited, the MuA recognition sites permit assembly of transpososomes in which non-Mu DNA substitutes for some of the Mu sequences. These "hybrid" transpososomes are stable to competitor DNA, actively transpose the non-Mu DNA, and produce transposition products that had been previously observed but not explained. The strongest activator of non-Mu transposition is a DNA fragment containing two MuA recognition sites and no cleavage site, but a shorter fragment with just one recognition site is sufficient. Based on our results, we propose that MuA recognition sites drive assembly of functional transpososomes in two complementary ways. Multiple recognition sites help physically position MuA subunits in the transpososome plus each individual site allosterically activates transposase.     

ClpX protein of Escherichia coli activates bacteriophage Mu transposase in the strand transfer complex for initiation of Mu DNA synthesis.

During transposition bacteriophage Mu transposase (MuA) catalyzes the transfer of a DNA strand at each Mu end to target DNA and then remains tightly bound to the Mu ends. Initiation of Mu DNA replication on the resulting strand transfer complex (STC1) requires specific host replication proteins and host factors from two partially purified enzyme fractions designated Mu replication factors alpha and beta (MRFalpha and beta). Escherichia coli ClpX protein, a molecular chaperone, is a component required for MRFalpha activity, which removes MuA from DNA for the establishment of a Mu replication fork. ClpX protein alters the conformation of DNA-bound MuA and converts STC1 to a less stable form (STC2). One or more additional components of MRFalpha (MRFalpha2) displace MuA from STC2 to form a nucleoprotein complex (STC3), that requires the specific replication proteins and MRFbeta for Mu DNA synthesis. MuA present in STC2 is essential for its conversion to STC3. If MuA is removed from STC2, Mu DNA synthesis no longer requires MRFalpha2, MRFbeta and the specific replication proteins. These results indicate that ClpX protein activates MuA in STC1 so that it can recruit crucial host factors needed to initiate Mu DNA synthesis by specific replication enzymes.     

Duplex opening by primosome protein PriA for replisome assembly on a recombination intermediate.

PriA and other primosome assembly proteins of Escherichia coli recruit the major replicative helicase DnaB for replisome assembly during bacteriophage Mu transposition and replication. MuA transposase catalyzes the transfer of Mu ends to target DNA, forming a potential replication fork that provides the assembly site for the replisome. However, this fork lacks the single-stranded DNA needed to load DnaB. Although no pre-existing primosome assembly sites that bind PriA were found within the Mu end sequences, PriA was able to bind to the forked DNA structure created by MuA. The helicase activity of PriA could then open the duplex to create the DnaB binding site. In a tightly coupled reaction on synthetic forked substrates, PriA promoted both the unwinding of the lagging strand arm and preprimosome assembly to load DnaB onto the lagging strand template. PriA apparently translocated 3' to 5' along the lagging strand template until sufficient single-stranded DNA was exposed for binding of DnaB, which then translocated 5' to 3' in the opposite direction. Mutant PriA lacking helicase activity was unable to promote this process, and loss of PriA helicase impaired Mu DNA replication in vivo and in vitro. This suggests that the opening of the duplex by PriA helicase is a critical step in the initiation of Mu DNA replication. Concerted helicase and primosome assembly functions would allow PriA to act as initiator on recombination intermediates and stalled replication forks. As part of the replisome, PriA may act as a mobile initiator that minimizes interruptions in chromosomal replication.     

Target immunity during Mu DNA transposition. Transpososome assembly and DNA looping enhance MuA-mediated disassembly of the MuB target complex

The Mu transpososome can distinguish between proximal and distal DNA during the selection of a site for transposition. This phenomenon, termed target immunity, involves MuA-stimulated removal of MuB oligomers from sites near the Mu genome. Using a combination of ensemble and single-molecule fluorescence methods, we show that the MuA tetramer can stably associate with the DNA-bound MuB oligomer and is more efficient than monomeric MuA at stimulating the dissociation of MuB from DNA. In addition, we demonstrate that DNA looping is essential for efficient disassembly of the MuB oligomer. We propose a model in which the MuA tetramer forms a multivalent complex with the MuB oligomer and catalyzes the processive removal of MuB from 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.     

Transposase contacts with mu DNA ends.

We have investigated the interaction between phage Mu transposase (A protein) and the ends (att sites) of Mu by chemical and nuclease protection and interference studies. These studies define a 24-base pair contact region at five of the six att sites (L1, L3 at att L and R1, R2, R3 at att R). Hydroxyl radical footprints show that the transposase binds to one face of the DNA helix and covers two consecutive major grooves. Binding specificity is achieved primarily through the major groove. Strong contacts are found with 3 guanines which are conserved at five of the sites. Two of these guanines are missing in the weakest binding site (L2) where 13 base pairs are mainly contacted. A pair of DNAase I hypersensitive sites, one on each strand, appear at the back of only one of the two contacted major grooves at most sites except at L2, and can be correlated with the degree of A protein-induced bend (Kuo, C.-F., Zou, A., Jayaram, M., Getzoff, E. D., and Harshey, R. M. (1991) EMBO J. 10, 1585-1591) at these sites. No contacts are observed for 4-5 base pairs in the vicinity of L1 and R1, where the A protein nicks DNA during transposition.     

Information analysis of Fis binding sites.

Originally discovered in the bacteriophage Mu DNA inversion system gin, Fis (Factor for Inversion Stimulation) regulates many genetic systems. To determine the base frequency conservation required for Fis to locate its binding sites, we collected a set of 60 experimentally defined wild-type Fis DNA binding sequences. The sequence logo for Fis binding sites showed the significance and likely kinds of base contacts, and these are consistent with available experimental data. Scanning with an information theory based weight matrix within fis, nrd, tgt/sec and gin revealed Fis sites not previously identified, but for which there are published footprinting and biochemical data. DNA mobility shift experiments showed that a site predicted to be 11 bases from the proximal Salmonella typhimurium hin site and a site predicted to be 7 bases from the proximal P1 cin site are bound by Fis in vitro. Two predicted sites separated by 11 bp found within the nrd promoter region, and one in the tgt/sec promoter, were also confirmed by gel shift analysis. A sequence in aldB previously reported to be a Fis site, for which information theory predicts no site, did not shift. These results demonstrate that information analysis is useful for predicting Fis DNA binding.     

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.     

Progressive structural transitions within Mu transpositional complexes

Assembly of the Mu transpososome is dependent on specific binding sites for the MuA transposase near the ends of the phage genome. MuA also contacts terminal nucleotides but only upon transpososome assembly, and base-specific recognition of the terminal nucleotides is critical for assembly. We show that Mu ends lacking the terminal 5 bp can form transpososomes, while longer DNA substrates with mutated terminal nucleotides cannot. The impact of the mutations can be suppressed by base mismatches near the end of Mu. Deletion of the flanking strands or mutation of the terminal nucleotides has differential effects on the cleavage and strand transfer reactions. These results show that the terminal nucleotides control the assembly and activation of transpososomes by influencing conformational changes around the active site.     

Sequence and positional requirements for DNA sites in a mu transpososome.

Transposition of bacteriophage Mu uses two DNA cleavage sites and six transposase recognition sites, with each recognition site divided into two half-sites. The recognition sites can activate transposition of non-Mu DNA sequences if a complete set of Mu sequences is not available. We have analyzed 18 sequences from a non-Mu DNA molecule, selected in a functional assay for the ability to be transposed by MuA transposase. These sequences are remarkably diverse. Nonetheless, when viewed as a group they resemble a Mu DNA end, with a cleavage site and a single recognition site. Analysis of these "pseudo-Mu ends" indicates that most positions in the cleavage and recognition sites contribute sequence-specific information that helps drive transposition, though only the strongest contributors are apparent from mutagenesis data. The sequence analysis also suggests variability in the alignment of recognition half-sites. Transposition assays of specifically designed DNA substrates support the conclusion that the transposition machinery is flexible enough to permit variability in half-site spacing and also perhaps variability in the placement of the recognition site with respect to the cleavage site. This variability causes only local perturbations in the protein-DNA complex, as indicated by experiments in which altered and unaltered DNA substrates are paired.     

Interactions of phage Mu enhancer and termini that specify the assembly of a topologically unique interwrapped transpososome

The higher-order DNA-protein complex that carries out the chemical steps of phage Mu transposition is organized by bridging interactions among three DNA sites, the left (L) and right (R) ends of Mu, and an enhancer element (E), mediated by the transposase protein MuA. A subset of the six subunits of MuA associated with their cognate sub-sites at L and R communicate with the enhancer to trigger the stepwise assembly of the functional transpososome. The DNA follows a well-defined path within the transpososome, trapping five supercoil nodes comprising two E-R crossings, one E-L crossing and two L-R crossings. The enhancer is a critical DNA element in specifying the unique interwrapped topology of the three-site LER synapse. In this study, we used multiple strategies to characterize Mu end-enhancer interactions to extend, modify and refine those inferred from earlier analyses. Directed placement of transposase subunits at their cognate sub-sites at L and R, analysis of the protein composition of transpososomes thus obtained, and their characterization using topological methods define the following interactions. R1-E interaction is essential to promote transpososome assembly, R3-E interaction contributes to the native topology of the transpososome, and L1-E and R2-E interactions are not required for assembly. The data on L2-E and L3-E interactions are not unequivocal. If they do occur, either one is sufficient to support the assembly process. Our results are consistent with two R-E and perhaps one L-E, being responsible for the three DNA crossings between the enhancer and the left and right ends of Mu. A 3D representation of the interwrapped complex (IW) obtained by modeling is consistent with these results. The model reveals straightforward geometric and topological relationships between the IW complex and a more relaxed enhancer-independent V-form of the transpososome assembled under altered reaction conditions.     

The Mu transposase interwraps distant DNA sites within a functional transpososome in the absence of DNA supercoiling

A Mu transpososome assembled on negatively supercoiled DNA traps five supercoils by intertwining the left (L) and right (R) ends of Mu with an enhancer element (E). To investigate the contribution of DNA supercoiling to this elaborate synapse in which E and L cross once, E and R twice, and L and R twice, we have analyzed DNA crossings in a transpososome assembled on nicked substrates under conditions that bypass the supercoiling requirement for transposition. We find that the transposase MuA can recreate an essentially similar topology on nicked substrates, interwrapping both E-R and L-R twice but being unable to generate the single E-L crossing. In addition, we deduce that the functional MuA tetramer must contribute to three of the four observed crossings and, thus, to restraining the enhancer within the complex. We discuss the contribution of both MuA and DNA supercoiling to the 5-noded Mu synapse built at the 3-way junction.