Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal.

The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal.

MuA-mediated in vitro cloning of circular DNA: transpositional autointegration and the effect of MuB.

Transposons provide useful tools for genetics and genomics studies, as they can be modified easily for a variety of purposes. In this study, a strategy to clone circular DNA was developed on the basis of an efficient Mu in vitro transposition reaction catalyzed by MuA transposase. The transposon used contains a selectable marker as well as an origin of replication, and in vitro integration of the transposon into circular DNA generates a plasmid that can replicate in E. coli. We show that the substrate stoichiometry plays an important role in the profile of intermolecular versus intramolecular transposition reaction products. Increasing the relative amount of target DNA reduced the frequency of intramolecular products that are non-productive with regard to the developed cloning application. Such autointegration was also reduced in the reactions containing phage Mu-encoded MuB, indicating that this protein can be used for cloning in combination with MuA, and it is particularly useful with a limited amount of target DNA. The developed strategy can now be utilized to clone DNA circles regardless of their origin as long as their size is not prohibitive for transformation.

Controlling DNA degradation from a distance: a new role for the Mu transposition enhancer.

Phage Mu is unique among transposable elements in employing a transposition enhancer. The enhancer DNA segment is the site where the transposase MuA binds and makes bridging interactions with the two Mu ends, interwrapping the ends with the enhancer in a complex topology essential for assembling a catalytically active transpososome. The enhancer is also the site at which regulatory proteins control divergent transcription of genes that determine the phage lysis-lysogeny decision. Here we report a third function for the enhancer - that of regulating degradation of extraneous DNA attached to both ends of infecting Mu. This DNA is protected from nucleases by a phage protein until Mu integrates into the host chromosome, after which it is rapidly degraded. We find that leftward transcription at the enhancer, expected to disrupt its topology within the transpososome, blocks degradation of this DNA. Disruption of the enhancer would lead to the loss or dislocation of two non-catalytic MuA subunits positioned in the transpososome by the enhancer. We provide several lines of support for this inference, and conclude that these subunits are important for activating degradation of the flanking DNA. This work also reveals a role for enhancer topology in phage development.

Unusual interaction of RNA polymerase with the bacteriophage Mu middle promoter Pm in the absence of its activator protein Mor.

The bacteriophage Mu Mor activator protein is absolutely required for transcription from the Mu middle promoter P(m). However, when RNA polymerase (RNAP) was incubated with P(m) DNA in the absence of Mor, a band at promoter position -51 was hypersensitive to DNase I cleavage, demonstrating an interaction of RNAP with the promoter DNA. The hypersensitivity was similar at four different lengths of P(m) DNA assayed from -62 to +10, -62 to +46, -96 to +10, and -96 to +46. The hypersensitivity occurred equally well at 5 °C, 15 °C, and 30 °C, indicating that it did not require open complex formation, which only occurred at 30 °C. The -51 hypersensitivity at 5 °C and 15 °C was eliminated by the addition of heparin, consistent with the possibility that it arose by formation of unstable closed complexes of RNAP bound to P(m) DNA. Generation of the hypersensitive band required the complete RNAP with its αCTDs, but neither the αCTD nor intact α were sufficient for the interaction and resulting hypersensitivity. There was no correlation between the level of hypersensitivity observed in vitro and the level of Pm activity in vivo, as assayed by the Mor-dependent production of ß-galactosidase from a P(m)-lacZ fusion. In an "order of addition" experiment, preincubation of P(m) DNA with Mor followed by addition of RNAP led to the fastest open complex formation, whereas preincubation of P(m) DNA with RNAP gave the slowest. These results support the conclusion that Mor recruits RNAP to P(m) rather than reposition a prebound RNAP, as occurs for C-dependent repositioning of RNAP at the Mu late promoter Pmom .

MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition.

MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.

The µ transpososome structure sheds light on DDE recombinase evolution.

Studies of bacteriophage Mu transposition paved the way for understanding retroviral integration and V(D)J recombination as well as many other DNA transposition reactions. Here we report the structure of the Mu transpososome--Mu transposase (MuA) in complex with bacteriophage DNA ends and target DNA--determined from data that extend anisotropically to 5.2 Å, 5.2 Å and 3.7 Å resolution, in conjunction with previously determined structures of individual domains. The highly intertwined structure illustrates why chemical activity depends on formation of the synaptic complex, and reveals that individual domains have different roles when bound to different sites. The structure also provides explanations for the increased stability of the final product complex and for its preferential recognition by the ATP-dependent unfoldase ClpX. Although MuA and many other recombinases share a structurally conserved 'DDE' catalytic domain, comparisons among the limited set of available complex structures indicate that some conserved features, such as catalysis in trans and target DNA bending, arose through convergent evolution because they are important for function.

Phage Mu transposition immunity: protein pattern formation along DNA by a diffusion-ratchet mechanism.

DNA transposons integrate into host chromosomes with limited target sequence specificity. Without mechanisms to avoid insertion into themselves, transposons risk self-destruction. Phage Mu avoids this problem by transposition immunity, involving MuA-transposase and MuB ATP-dependent DNA-binding protein. MuB-bound DNA acts as an efficient transposition target, but MuA clusters bound to Mu DNA ends activate the MuB-ATPase and dissociate MuB from their neighborhood before target site commitment, making the regions near Mu ends a poor target. This MuA-cluster-MuB interaction requires formation of DNA loops between the MuA- and the MuB-bound DNA sites. At early times, MuB clusters are disassembled via loops with smaller average size, and at later times, MuA clusters find distantly located MuB clusters by forming loops with larger average sizes. We demonstrate that iterative loop formation/disruption cycles with intervening diffusional steps result in larger DNA loops, leading to preferential insertion of the transposon at sites distant from the transposon ends.

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.

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.

Dissection of the bacteriophage Mu strong gyrase site (SGS): significance of the SGS right arm in Mu biology and DNA gyrase mechanism.

The bacteriophage Mu strong gyrase site (SGS), required for efficient phage DNA replication, differs from other gyrase sites in the efficiency of gyrase binding coupled with a highly processive supercoiling activity. Genetic studies have implicated the right arm of the SGS as a key structural feature for promoting rapid Mu replication. Here, we show that deletion of the distal portion of the right arm abolishes efficient binding, cleavage, and supercoiling by DNA gyrase in vitro. DNase I footprinting analysis of the intact SGS revealed an adenylyl imidodiphosphate-dependent change in protection in the right arm, indicating that this arm likely forms the T segment that is passed through the cleaved G segment during the supercoiling reaction. Furthermore, in an SGS derivative with an altered right-arm sequence, the left arm showed these changes, suggesting that the selection of a T segment by gyrase is determined primarily by the sequences of the arms. Analysis of the sequences of the SGS and other gyrase sites suggests that the choice of T segment correlates with which arm possesses the more extensive set of phased anisotropic bending signals, with the Mu right arm possessing an unusually extended set of such signals. The implications of these observations for the structure of the gyrase-DNA complex and for the biological function of the Mu SGS are discussed.

3D reconstruction of the Mu transposase and the Type 1 transpososome: a structural framework for Mu DNA transposition.

Mu DNA transposition proceeds through a series of higher-order nucleoprotein complexes called transpososomes. The structural core of the transpososome is a tetramer of the transposase, Mu A, bound to the two transposon ends. High-resolution structural analysis of the intact transposase and the transpososome has not been successful to date. Here we report the structure of Mu A at 16-angstroms and the Type 1 transpososome at 34-angstroms resolution, by 3D reconstruction of images obtained by scanning transmission electron microscopy (STEM) at cryo-temperatures. Electron spectroscopic imaging (ESI) of the DNA-phosphorus was performed in conjunction with the structural investigation to derive the path of the DNA through the transpososome and to define the DNA-binding surface in the transposase. Our model of the transpososome fits well with the accumulated biochemical literature for this intricate transposition system, and lays a structural foundation for biochemical function, including catalysis in trans and the complex circuit of macromolecular interactions underlying Mu DNA transposition.

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.

Characterization of Plys-proximal morphogenetic genes of transposable bacteriophage Mu.

Late during the bacteriophage Mu lytic cycle, Mu DNA must be matured and packaged from its dispersed integration sites in the host DNA in order to produce progeny virions. Whereas control of late gene transcription in Mu is becoming well understood, less is known about the phage morphogenetic process. To investigate the latter, we cloned and sequenced a approximately 4.3-kb region of the phage DNA beginning just upstream of the leftmost late promoter Plys. Previous mapping of amber mutations had located the lysis (lys) and proposed DNA maturation genes D and E in this region. When the DNA sequence was analyzed, seven potential open reading frames were found. DNA sequence analysis of amber mutations in genes D and E identified the sixth and seventh open reading frames as D and E, respectively. Cloning and expression of this region enabled production of cell-free protein extracts that specifically recognize the phage-encoded packaging sequence (pac), a characteristic exhibited by phage maturation enzymes. In addition, the E protein was found to share homology with the large subunit of many phage DNA maturation enzymes. These results support the hypothesis that D and E encode subunits of the Mu DNA maturation enzyme.

MuA transposase separates DNA sequence recognition from catalysis.

Confronted with thousands of potential DNA substrates, a site-specific enzyme must restrict itself to the correct DNA sequence. The MuA transposase protein performs site-specific DNA cleavage and joining reactions, resulting in DNA transposition-a specialized form of genetic recombination. To determine how sequence information is used to restrict transposition to the proper DNA sites, we performed kinetic analyses of transposition with DNA substrates containing either wild-type transposon sequences or sequences carrying mutations in specific DNA recognition modules. As expected, mutations near the DNA cleavage site reduce the rate of cleavage; the observed effect is about 10-fold. In contrast, mutations within the MuA recognition sequences do not directly affect the DNA cleavage or joining steps of transposition. It is well established that the recognition sequences are necessary for assembly of stable, multimeric MuA-DNA complexes, and we find that recognition site mutations severely reduce both the extent and the rate of this assembly process. Yet if the MuA-DNA complexes are preassembled, the first-order rate constants for both DNA cleavage and DNA strand transfer (the joining reaction) are unaffected by the mutations. Furthermore, most of the mutant DNA molecules that are cleaved also complete DNA strand transfer. We conclude that the sequence-specific contacts within the recognition sites contribute energetically to complex assembly, but not directly to catalysis. These results contrast with studies of more orthodox enzymes, such as EcoRI and some other type II restriction enzymes. We propose that the strategy employed by MuA may serve as an example for how recombinases and modular restriction enzymes solve the DNA specificity problem, in that they, too, may separate substrate recognition from catalysis.

Mu transpososome architecture ensures that unfolding by ClpX or proteolysis by ClpXP remodels but does not destroy the complex.

The Clp/Hsp100 ATPases are protein unfoldases that both alter protein conformation and target proteins for degradation. An unresolved question has been how such seemingly destructive enzymes can "remodel" some protein substrates rather than destroy them. Here, we investigate the products of ClpX-mediated remodeling of a hyper-stable protein-DNA complex, the Mu transpososome. We find that although an oligomeric complex is maintained, release of some subunits accompanies ClpX action. Replacement of transposase's endogenous ClpX-recognition sequence with an exogenous signal reveals that the mechanism of remodeling is independent of both the recognition signal and the identity of the unfoldase. Finally, examination of the transposase-DNA contacts reveals only a localized region that is altered during remodeling. These results provide a framework for protein remodeling, wherein the physical attributes of a complex can limit the unfolding activity of its remodeler.

Identification and characterization of an endolysin encoded by the Streptomyces aureofaciens phage mu 1/6.

An open reading frame homologous to the genes encoding several cell-wall hydrolyzing enzymes was identified on the genome of actinophage mu 1/6. This open reading frame encoding the putative endolysin was amplified by polymerase chain reaction and cloned into the expression vector pET-21a. This gene consisted of 1182 bp encoding a 393 amino acid polypeptide with a molar mass of 42.1 kDa. The gene product was overexpressed in Escherichia coli, and then the lytic enzyme was purified by a two-step chromatographic procedure. When applied exogenously, the endolysin of phage mu 1/6 was active against all tested Streptomyces strains but did not affect other bacteria. The amino acid sequence showed a high homology with a putative amidase of the Streptomyces phase phi C31. Downstream of the endolysin gene, an open reading frame encoding an 88 amino acid protein was identified. Structural analysis of its sequence revealed features characteristics for holin.

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.

The Mu repressor-DNA complex contains an immobilized 'wing' within the minor groove.

We have determined the solution structure of the complex between the 'winged-helix' enhancer binding domain of the Mu repressor protein and its cognate DNA site. The structure reveals an unusual use for the 'wing' which becomes immobilized upon DNA binding where it makes intermolecular hydrogen bond contacts deep within the minor groove. Although the wing is mobile in the absence of DNA, it partially negates the large entropic penalty associated with its burial by maintaining a small degree of structural order in the DNA-free state. Extensive contacts are also formed between the recognition helix and the DNA, which reads the major groove of a highly conserved region of the binding site through a single base-specific hydrogen bond and van der Waals contacts.

Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replication.

The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini. Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication. To determine the features required for its unique properties, a deletion analysis was performed on the SGS. For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the 'core' and the flanking sequences to be the 'arms'. The deletion analysis showed that (i) approximately 40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage. These observations implicated the right arm as the unique feature of the SGS. The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication. Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication. The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site. The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication. Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences. Possible roles for the SGS right arm in Mu replication are addressed.