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.     

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.     

Effect of mutations in the Mu-host junction region on transpososome assembly.

Mu transposition occurs through a series of higher-order nucleoprotein complexes called transpososomes. The region where the Mu DNA joins the host DNA plays an integral role in the assembly of these transpososomes. We have created a series of point mutations at the Mu-host junction and characterized their effect on the Mu in vitro strand transfer reaction. Analysis of these mutant constructs revealed an inhibition in transpososome assembly at the point in the reaction pathway when the junction region is engaged by the transposase active site (i.e. the transition from LER to type 0). We found that the degree of inhibition was dependent upon the particular base-pair change at each position and whether the substitution occurred at the left or right transposon end. The MuB transposition protein, an allosteric effector of MuA, was shown to suppress all of the inhibitory Mu-host junction mutants. Most of the mutant constructs were also suppressed, to varying degrees, by the substitution of Mg(2+) with Mn(2+). Analysis of the mutant constructs has revealed hierarchical nucleotide preferences at positions -1 through +3 for transpososome assembly and suggests the possibility that specific metal ion-DNA base interactions are involved in DNA recognition and transpososome assembly.     

Reorganization of the Mu transpososome active sites during a cooperative transition between DNA cleavage and joining

Transposition of mobile genetic elements proceeds through a series of DNA phosphoryl transfer reactions, with multiple reaction steps catalyzed by the same set of active site residues. Mu transposase repeatedly utilizes the same active site DDE residues to cleave and join a single DNA strand at each transposon end to a new, distant DNA location (the target DNA). To better understand how DNA is manipulated within the Mu transposase-DNA complex during recombination, the impact of the DNA immediately adjacent to the Mu DNA ends (the flanking DNA) on the progress of transposition was investigated. We show that, in the absence of the MuB activator, the 3 '-flanking strand can slow one or more steps between DNA cleavage and joining. The presence of this flanking DNA strand in just one active site slows the joining step in both active sites. Further evidence suggests that this slow step is not due to a change in the affinity of the transpososome for the target DNA. Finally, we demonstrate that MuB activates transposition by stimulating the reaction step between cleavage and joining that is otherwise slowed by this flanking DNA strand. Based on these results, we propose that the 3 '-flanking DNA strand must be removed from, or shifted within, both active sites after the cleavage step; this movement is coupled to a conformational change within the transpososome that properly positions the target DNA simultaneously within both active sites and thereby permits joining.     

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.     

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.     

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.     

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 phage Mu transpososome core: DNA requirements for assembly and function.

The two chemical steps of phage Mu transpositional recombination, donor DNA cleavage and strand transfer, take place within higher order protein-DNA complexes called transpososomes. At the core of these complexes is a tetramer of MuA (the transposase), bound to the two ends of the Mu genome. While transpososome assembly normally requires a number of cofactors, under certain conditions only MuA and a short DNA fragment are required. DNA requirements for this process, as well as the stability and activity of the ensuing complexes, were established. The divalent cation normally required for assembly of the stable complex could be omitted if the substrate was prenicked, if the flanking DNA was very short or if the two flanking strands were non-complementary. The presence of a single nucleotide beyond the Mu genome end on the non-cut strand was critical for transpososome stability. Donor cleavage additionally required at least two flanking nucleotides on the strand to be cleaved. The flanking DNA double helix was destabilized, implying distortion of the DNA near the active site. Although donor cleavage required Mg2+, strand transfer took place in the presence of Ca2+ as well, suggesting a conformational difference in the active site for the two chemical steps.     

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.     

A unique right end-enhancer complex precedes synapsis of Mu ends: the enhancer is sequestered within the transpososome throughout transposition

Assembly of the Mu transpososome is dependent on interactions of transposase subunits with the left (L) and right (R) ends of Mu and an enhancer (E). We have followed the order and dynamics of association of these sites within a series of transpososomes prior to and during formation of a three-site complex (LER), engagement of Mu ends by the transposase active site (type 0 complex), cleavage of the ends (type I complex) and their transfer to target DNA (type II complex). LER appears to be preceded by a two-site complex (ER) where E and R are interwrapped twice, as in the mature transpososome. At each stage thereafter, the overall topology of five DNA supercoils is retained: two between E and R, one between E and L and two between L and R. However, L-R interactions within LER appear to be flexible. Unexpectedly, the enhancer was seen to persist within the transpososome through cleavage and strand transfer of Mu ends to target DNA.     

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.     

An ATP-ADP switch in MuB controls progression of the Mu transposition pathway.

MuB protein, an ATP-dependent DNA-binding protein, collaborates with Mu transposase to promote efficient transposition. MuB binds target DNA, delivers this target DNA segment to transposase and activates transposase''s catalytic functions. Using ATP-bound, ADP-bound and ATPase-defective MuB proteins we investigated how nucleotide binding and hydrolysis control the activities of MuB protein, important for transposition. We found that both MuB-ADP and MuB-ATP stimulate transposase, whereas only MuB-ATP binds with high affinity to DNA. Four different ATPase-defective MuB mutants fail to activate the normal transposition pathway, further indicating that ATP plays critical regulatory roles during transposition. These mutant proteins fall into two classes: class I mutants are defective in target DNA binding, whereas class II mutants bind target DNA, deliver it to transposase, but fail to promote recombination with this DNA. Based on these studies, we propose that the switch from the ATP- to ADP-bound form allows MuB to release the target DNA while maintaining its stimulatory interaction with transposase. Thus, ATP-hydrolysis by MuB appears to function as a molecular switch controlling how target DNA is delivered to the core transposition machinery.     

Altering the DNA-binding specificity of Mu transposase in vitro.

We describe the isolation of a variant of Mu transposase (MuA protein) which can recognize altered att sites at the ends of Mu DNA. No prior knowledge of the structure of the DNA binding domain or its mode of interaction with att DNA was necessary to obtain this variant. Protein secondary structure programs initially helped target mutations to predicted helical regions within a subdomain of MuA demonstrated to harbor att DNA binding activity. Of the 54 mutant positions examined, only two showed decreased affinity for att DNA, while eight others affected assembly of the Mu transpososome. A variant impaired in DNA binding [MuA(R146V)], and predicted to be in the recognition helix of an HTH motif, was challenged with altered att sites created from degenerate oligonucleotides to select for novel DNA binding specificity. DNA sequences bound to MuA(R146V) were detected by gel-retardation, and following several steps of PCR amplification/enrichment, were identified by cloning and sequencing. The strategy allowed recovery of an altered att site for which MuA(R146V) showed higher affinity than for the wild-type site, although this site was bound by wild-type MuA as well. The altered association between MuA(R146V) and an altered att site target was competent in transposition. We discuss the strengths and limitations of this methodology, which has applications in dissecting the functional role of specific protein-DNA associations.     

Flexibility in MuA transposase family protein structures: functional mapping with scanning mutagenesis and sequence alignment of protein homologues

MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.     

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.     

The Mu three-site synapse: a strained assembly platform in which delivery of the L1 transposase binding site triggers catalytic commitment

The Mu DNA transposition reaction proceeds through a three-site synaptic complex (LER), including the two Mu ends and the transpositional enhancer. We show that the LER contains highly stressed DNA regions in the enhancer and in the L1 transposase binding site. We propose that the L1 site acts as the keystone for assembly of a catalytically competent transpososome. Delivery of L1 through HU-mediated bending completes LER assembly, provides the trigger for necessary conformational transitions in transpososome formation, and allows target capture to occur. Relief of the stress at L1 and the enhancer may help drive Mu A tetramerization and engagement of the Mu ends by the transposase active site.     

IHF-independent assembly of the Tn10 strand transfer transpososome: implications for inhibition of disintegration

The frequency of DNA transposition in transposition systems that employ a strand transfer step may be significantly affected by the occurrence of a disintegration reaction, a reaction that reverses the strand transfer event. We have asked whether disintegration occurs in the Tn10 transposition system. We show that disintegration substrates (substrates constituting one half of the strand transfer product) are assembled into a transpososome that mimics the strand transfer intermediate. This strand transfer transpososome (STT) does appear to support an intermolecular disintegration reaction, but only at a very low level. Strikingly, assembly of the STT is not dependent on IHF, a host protein that is required for de novo assembly of all previously characterized Tn10 transpososomes. We suggest that disintegration substrates are able to form both transposon end and target type contacts with transposase because of their enhanced conformational flexibility. This probably allows the conformation of DNA within the complex that prevents the destructive disintegration reaction, and is responsible for relaxing the DNA sequence requirements for STT formation relative to other Tn10 transpososomes.     

Characteristics of MuA transposase-catalyzed processing of model transposon end DNA hairpin substrates

Bacteriophage Mu uses non-replicative transposition for integration into the host's chromosome and replicative transposition for phage propagation. Biochemical and structural comparisons together with evolutionary considerations suggest that the Mu transposition machinery might share functional similarities with machineries of the systems that are known to employ a hairpin intermediate during the catalytic steps of transposition. Model transposon end DNA hairpin substrates were used in a minimal-component in vitro system to study their proficiency to promote Mu transpososome assembly and subsequent MuA-catalyzed chemical reactions leading to the strand transfer product. MuA indeed was able to assemble hairpin substrates into a catalytically competent transpososome, open the hairpin ends and accurately join the opened ends to the target DNA. The hairpin opening and transposon end cleavage reactions had identical metal ion preferences, indicating similar conformations within the catalytic center for these reactions. Hairpin length influenced transpososome assembly as well as catalysis: longer loops were more efficient in these respects. In general, MuA's proficiency to utilize different types of hairpin substrates indicates a certain degree of flexibility within the transposition machinery core. Overall, the results suggest that non-replicative and replicative transposition systems may structurally and evolutionarily be more closely linked than anticipated previously.