Mu End Research Articles

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA–DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein–protein and protein–DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

Transposable Phage Mu.

Transposable phage Mu has played a major role in elucidating the mechanism of movement of mobile DNA elements. The high efficiency of Mu transposition has facilitated a detailed biochemical dissection of the reaction mechanism, as well as of protein and DNA elements that regulate transpososome assembly and function. The deduced phosphotransfer mechanism involves in-line orientation of metal ion-activated hydroxyl groups for nucleophilic attack on reactive diester bonds, a mechanism that appears to be used by all transposable elements examined to date. A crystal structure of the Mu transpososome is available. Mu differs from all other transposable elements in encoding unique adaptations that promote its viral lifestyle. These adaptations include multiple DNA (enhancer, SGS) and protein (MuB, HU, IHF) elements that enable efficient Mu end synapsis, efficient target capture, low target specificity, immunity to transposition near or into itself, and efficient mechanisms for recruiting host repair and replication machineries to resolve transposition intermediates. MuB has multiple functions, including target capture and immunity. The SGS element promotes gyrase-mediated Mu end synapsis, and the enhancer, aided by HU and IHF, participates in directing a unique topological architecture of the Mu synapse. The function of these DNA and protein elements is important during both lysogenic and lytic phases. Enhancer properties have been exploited in the design of mini-Mu vectors for genetic engineering. Mu ends assembled into active transpososomes have been delivered directly into bacterial, yeast, and human genomes, where they integrate efficiently, and may prove useful for gene therapy.

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.

WE-E-AUD-05: Dosimetric Characteristics of GAFCHROMIC® EBT Film for Therapeutic Electron Beams

Purpose: To evaluate the dosimetric characteristics of GAFCHROMIC® EBT film when exposed by high‐energy electron beams.Method and Materials: Studies of the effects of the electron beam treatment parameters on dose calibration curves were performed with GAFCHROMIC® EBT radiochromic films. All films used in this study were from the same batch in order to remove any variability between batches. The films were irradiated in a multilayered 30 × 30 × 25 cm3 solid water phantom with source‐to‐surface (SSD) distance of 100 cm. The effects of electron beam energy, cone size, dose rate, depth, and MU end effect were studied. Results: The dose response curve was found to be independent of the electron energy (−2.2% to 3.6% difference of the averaged scan value). The dose calibration curves of EBT films were independent of MU end effect (from −1.0% to 2.2%) as well as electron beam dose rate (within 1.7%). For the field size dependence due to different electron cones, it is possible to calibrate GAFCHROMIC® EBT films using 10 × 10 cm2, cone size and then evaluate dose distributions resulting from various electron cone sizes. The EBT film did not show any impact of the depth on the dose response curve for electron beams. Conclusions: There was no much influence on dose response curves of GAFCHROMIC® EBT films due to electron beam energy, cone size, dose rate, depth, and MU end effect. The independence of the GAFCHROMIC® EBT on these electron beam dosimetric parameters studied for this kind of newly developed radiochromic film makes it an accurate and versatile dosimetric detector for two‐dimensional dose distribution measurements for therapeuticelectron beams.

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.

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.

Versatile Action of Escherichia coli ClpXP as Protease or Molecular Chaperone for Bacteriophage Mu Transposition

The molecular chaperone ClpX of Escherichia coli plays two distinct functions for bacteriophage Mu DNA replication by transposition. As specificity component of a chaperone-linked protease, it recognizes the Mu immunity repressor for degradation by the peptidase component ClpP, thus derepressing Mu transposition functions. After strand exchange has been promoted by MuA transposase, ClpX alone can alter the conformation of the transpososome (the complex of MuA with Mu ends), and the remodeled MuA promotes transition to replisome assembly. Although ClpXP can degrade MuA, the presence of both ClpP and ClpX in the reconstituted transposition system did not destroy MuA essential for initiation of DNA replication by specific host replication enzymes. Levels of ClpXP needed to overcome inhibition by the repressor did not prevent MuA from promoting strand transfer, and ClpP stimulated alteration of the transpososome by ClpX. Apparently intact MuA was still present in the resulting transpososome, promoting initiation of Mu DNA replication by specific replication enzymes. The results indicate that ClpXP can discriminate repressor and MuA in the transpososome as substrates of the protease or the molecular chaperone alone, degrading repressor while remodeling MuA for its next critical function.

Solution structure of the Mu end DNA-binding ibeta subdomain of phage Mu transposase: modular DNA recognition by two tethered domains.

The phage Mu transposase (MuA) binds to the ends of the Mu genome during the assembly of higher order nucleoprotein complexes. We investigate the structure and function of the MuA end-binding domain (Ibetagamma). The three-dimensional solution structure of the Ibeta subdomain (residues 77-174) has been determined using multidimensional NMR spectroscopy. It comprises five alpha-helices, including a helix-turn-helix (HTH) DNA-binding motif formed by helices 3 and 4, and can be subdivided into two interacting structural elements. The structure has an elongated disc-like appearance from which protrudes the recognition helix of the HTH motif. The topology of helices 2-4 is very similar to that of helices 1-3 of the previously determined solution structure of the MuA Igamma subdomain and to that of the homeodomain family of HTH DNA-binding proteins. We show that each of the two subdomains binds to one half of the 22 bp recognition sequence, Ibeta to the more conserved Mu end distal half (beta subsite) and Igamma to the Mu end proximal half (gamma subsite) of the consensus Mu end-binding site. The complete Ibetagamma domain binds the recognition sequence with a 100- to 1000-fold higher affinity than the two subdomains independently, indicating a cooperative effect. Our results show that the Mu end DNA-binding domain of MuA has a modular organization, with each module acting on a specific part of the 22 bp binding site. Based on the present binding data and the structures of the Ibeta and Igamma subdomains, a model for the interaction of the complete Ibetagamma domain with DNA is proposed.

Solution structure of the Iγ subdomain of the Mu end DNA-binding domain of phage Mu transposase

The MuA transposase of phase Mu is a large modular protein that plays a central role in transposition. We show that the Mu end DNA-binding domain, Iβγ, which is responsible for binding the DNA attachment sites at each end of the Mu genome, comprises two subdomains, Iβ and Iγ, that are structurally autonomous and do not interact with each other in the absence of DNA. The solution structure of the Iγ subdomain has been determined by multidimensional NMR spectroscopy. The structure of Iγ comprises a four helix bundle and, despite the absence of any significant sequence identity, the topology of the first three helices is very similar to that of the homeodomain family of helix-turn-helix DNA-binding proteins. The helix-turn-helix motif of Iγ, however, differs from that of the homeodomains in so far as the loop is longer and the second helix is shorter, reminiscent of that in the POU-specific domain.

Kinetic and Structural Probing of the Precleavage Synaptic Complex (Type 0) Formed during Phage Mu Transposition: ACTION OF METAL IONS AND REAGENTS SPECIFIC TO SINGLE-STRANDED DNA

In an earlier kinetic study (Wang, Z., and Harshey, R. M. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 699-703), we showed that supercoiling free energy was utilized during Mu transposition to lower the activation barrier of some rate-limiting step in the formation of the cleaved Mu end synaptic complex (type I complex). We report here results from kinetic studies on the assembled but uncleaved synaptic complex (type 0). Based on the estimated rate constants for the formation of type 0 and type I complexes, as well as their temperature and superhelicity dependence, we infer that the type 0 complex is an authentic intermediate in the pathway to Mu end cleavage. Our results are consistent with type 0 production being the rate-limiting step in the overall type I reaction. The conversion of type 0 to type I complex is a fast reaction, does not show strong temperature dependence, and is apparently independent of substrate superhelicity. We have explored the DNA structure within the type 0 complex using chemical and enzymatic probes. The observed susceptibility of DNA outside the Mu ends to single-strand-specific reagents suggests that a helix opening event is associated with type 0 formation. This structural perturbation could account, at least partly, for the high activation barrier to the reaction. There is a close correlation between the appearance of single strandedness near the Mu ends and the superhelicity of the DNA substrate. It is possible that supercoiling energy is utilized in effecting specific conformational transitions within DNA. We have found that Zn2+ and Co2+ ions, like Mg2+ and Mn2+ ions, can efficiently cleave the type 0 complex. However, unlike Mg2+ and Mn2+ ions, Zn2+ and Co2+ ions cannot support assembly of type 0. We discuss the implications of our findings for the mechanism of Mu transposition.

Mu Transpositional Recombination: Donor DNA Cleavage and Strand Transfer in trans by the Mu Transposase

Central to the Mu transpositional recombination are the two chemical steps; donor DNA cleavage and strand transfer. These reactions occur within the Mu transpososome that contains two Mu DNA end segments bound to a tetramer of MuA, the transposase. To investigate which MuA monomer catalyzes which chemical reaction, we made transpososomes containing wild-type and active site mutant MuA. By preloading the MuA variants onto Mu end DNA fragments of different length prior to transpososome assembly, we could track the catalysis by MuA bound to each Mu end segment. The donor DNA end that underwent the chemical reaction was identified. Both the donor DNA cleavage and strand transfer were catalyzed in trans by the MuA monomers bound to the partner Mu end. This arrangement explains why the transpososome assembly is a prerequisite for the chemical steps.

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.

A domain sharing model for active site assembly within the Mu A tetramer during transposition: the enhancer may specify domain contributions.

The functional configuration of Mu transposase (A protein) is its tetrameric form. We present here a model for the organization of a functional Mu A tetramer. Within the tetramer, assembly of each of the two active sites for Mu end cleavage requires amino acid contributions from the central and C-terminal domains (domains II and III respectively) of at least two Mu A monomers in a trans configuration. The Mu enhancer is likely to function in this assembly process by specifying the two monomers that provide their C-terminal domains for strand cleavage. The Mu B protein is not required in this step. Each of the two active sites for the strand transfer reaction is also organized by domain sharing (but in the reverse mode) between Mu A monomers; i.e. a donor of domain II (also the recipient of domain III) during cleavage is a recipient of domain II (and the donor of domain III) during strand transfer. The function of the Mu B protein (which is required at the strand transfer step) and that of the enhancer element may be analogous in that their interactions with Mu A (domain III and domain I alpha respectively) promote conformations of Mu A conducive to strand cleavage or strand transfer.

Step-arrest Mutants of Phage Mu Transposase: IMPLICATIONS IN DNA-PROTEIN ASSEMBLY, Mu END CLEAVAGE, AND STRAND TRANSFER

We describe the isolation and characterization of Mu A variants arrested at specific steps of transposition. Mutations at 13 residues within the Mu A protein were analyzed for precise excision of Mu DNA in vivo. A subset of the defective variants (altered at Asp269, Asp294, Gly348, and Glu392) were tested in specific steps of transposition in vitro. It is possible that at least some residues of the Asp269-Asp294-Glu392 triad may have functional similarities to those of the conserved Asp-Asp-Glu motif found in several transposases and retroviral integrases. Mu A(D269V) is defective in high-order DNA-protein assembly, Mu end cleavage, and strand transfer. The assembly defect, but not the catalytic defect, can be overcome by precleavage of Mu ends. Mu A(E392A) can assemble the synaptic complex, but cannot cleave Mu ends. A mutation of Gly348 to aspartic acid within Mu A permits the uncoupling of cleavage and strand transfer activities. This mutant is completely defective in synaptic assembly and Mu end cleavage in presence of Mg2+. The assembly defect is alleviated by replacing Mg2+ with Ca2+. Some Mu end cleavage is observed with this mutant in the presence of Mn2+. When presented with precleaved Mu ends, Mu A(G348D) exhibits efficient strand transfer activity.

A new component of bacteriophage Mu replicative transposition machinery: the Escherichia coli ClpX protein

We have shown previously that some particular mutations in bacteriophage Mu repressor, the frameshift vir mutations, made the protein very sensitive to the Escherichia coli ATP-dependent Clp protease. This enzyme is formed by the association between a protease subunit (ClpP) and an ATPase subunit. ClpA, the best characterized of these ATPases, is not required for the degradation of the mutant Mu repressors. Recently, a new potential ClpP associated ATPase, ClpX, has been described. We show here that this new subunit is required for Mu vir repressor degradation. Moreover, ClpX (but not ClpP) was found to be required for normal Mu replication. Thus ClpX has activities that do not require its association with ClpP. In the pathway of Mu replicative transposition, the block resides beyond the strand transfer reaction, i.e. after the transposition reaction per se is completed, suggesting that ClpX is required for the transition to the formation of the active replication complex at one Mu end. This is a new clear-cut case of the versatile activity of polypeptides that form multi-component ATP-dependent proteases.

DNase protection analysis of the stable synaptic complexes involved in Mu transposition.

Several critical steps in phage Mu transposition involve specialized protein-DNA complexes. Cleavage of Mu donor DNA by MuA protein leads to the formation of the stable cleaved donor complex (CDC), in which the two Mu DNA ends are held together by MuA. In the subsequent strand-transfer reaction the CDC attacks a target DNA to generate the strand-transfer complex, in which the donor and the target DNAs are covalently joined. We have carried out DNase I protection experiments on these protein-DNA complexes and found that only three MuA binding sites (L1, R1, and R2 of the six total) at the two Mu ends are stably bound by MuA to maintain the paired Mu end structure. The protection extends beyond the ends of the Mu sequence for different lengths (7-20 nucleotides) depending on the strand and the type of complex. After formation of the CDC, the other MuA binding sites (L2, L3, and R3) and internal activation sequence become dispensable for the subsequent strand-transfer reaction.

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.

Efficient Mu transposition requires interaction of transposase with a DNA sequence at the Mu operator: Implications for regulation

Phage Mu transposition is initiated by the Mu DNA strand-transfer reaction, which generates a branched DNA structure that acts as a transposition intermediate. A critical step in this reaction is formation of a special synaptic DNA-protein complex called a plectosome. We find that formation of this complex involves, in addition to a pair of Mu end sequences, a third cis-acting sequence element, the internal activation sequence (IAS). The IAS is specifically recognized by the N-terminal domain of Mu transposase (MuA protein). Neither the N-terminal domain of MuA protein nor the IAS is required for later reaction steps. The IAS overlaps with the sequences to which Mu repressor protein binds in the Mu operator region; the Mu repressor directly inhibits the Mu DNA strand-transfer reaction by interfering with the interaction between MuA protein and the IAS, providing an additional mode of regulation by the repressor.

Interaction of proteins located at a distance along DNA: Mechanism of target immunity in the Mu DNA strand-transfer reaction

DNA molecules carrying a Mu end(s) are inefficient targets in the Mu DNA strand-transfer reaction. This target immunity is due to preferential dissociation of Mu B protein from DNA molecules that have Mu A protein bound to the Mu end; free DNA is a much poorer target than DNA with Mu B protein bound. We show that Mu B protein, which binds nonspecifically to DNA, is immobile once bound. An encounter between Mu A and Mu B proteins, bound some distance apart along DNA, is necessary to facilitate the Mu B dissociation. Experiments which show that DNA without a Mu end can acquire immunity, by catenation to DNA with a Mu end(s), are consistent with a model of Mu A-Mu B interaction by DNA looping, but not by linear movement of protein(s) along DNA.

Target immunity of Mu transposition reflects a differential distribution of Mu B protein

A DNA molecule carrying Mu end DNA sequence(s) is a poor target in the Mu DNA strand-transfer reaction, a phenomenon which is referred to as “target immunity.” We find that Mu B protein stimulates intermolecular strand-transfer by binding to the target DNA. Our results show that a differential distribution of Mu B protein between “immune” and “non-immune” DNA molecules is responsible for target immunity; in the presence of Mu A protein and ATP, Mu B protein dissociates preferentially from immune DNA molecules. Hydrolysis of ATP is implicated in establishing the differential distribution of Mu B protein between immune and non-immune DNA molecules in the presence of Mu A protein; nonhydrolyzable ATPγ can support an efficient strand-transfer reaction even with a target DNA that is immune in a reaction with ATP.