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.     

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.     

DNA gyrase requirements distinguish the alternate pathways of Mu transposition

The MuA transposase mediates transposition of bacteriophage Mu through two distinct mechanisms. The first integration event following infection occurs through a non-replicative mechanism. In contrast, during lytic growth, multiple rounds of replicative transposition amplify the phage genome. We have examined the influence of gyrase and DNA supercoiling on these two transposition pathways using both a gyrase-inhibiting drug and several distinct gyrase mutants. These experiments reveal that gyrase activity is not essential for integration; both lysogens and recombination intermediates are detected when gyrase is inhibited during Mu infection. In contrast, gyrase inhibition causes severe defects in replicative transposition. In two of the mutants, as well as in drug-treated cells, replicative transposition is almost completely blocked. Experiments probing for formation of MuA-DNA complexes in vivo reveal that this block occurs very early, during assembly of the transposase complex required for the catalytic steps of recombination. The findings establish that DNA structure-based signals are used differently for integrative and replicative transposition. We propose that transposase assembly, the committed step for recombination, has evolved to depend on different DNA /architectural signals to control the reaction outcome during these two distinct phases of the phage life cycle.     

Mechanism of transposition of bacteriophage Mu: structure of a transposition intermediate

Mu transposition works efficiently in vitro and generates both cointegrate and simple insert products. We have examined the reaction products obtained under modified in vitro reaction conditions that do not permit efficient initiation of DNA replication. The major product is precisely the intermediate structure predicted from one of the current models of DNA transposition. Both cointegrates and simple inserts can be made in vitro using this intermediate as the DNA substrate, demonstrating that it is indeed a true transposition intermediate. The requirements for efficient formation of the intermediate include the Mu A protein, the Mu B protein, an unknown number of E. coli host proteins, ATP, and divalent cation. Only E. coli host proteins are required for conversion of the intermediate to cointegrate or simple insert products. Structures resulting from DNA strand transfer at only one end of the transposon are not observed, suggesting that the strand transfers at each end of the transposon are tightly coupled.     

A new set of Mu DNA transposition intermediates: alternate pathways of target capture preceding strand transfer.

Mu DNA transposition occurs within the context of higher order nucleoprotein structures or transpososomes. We describe a new set of transpososomes in which Mu B-bound target DNA interacts non-covalently with previously characterized intermediates prior to the actual strand transfer. This interaction can occur at several points along the reaction pathway: with the LER, the Type 0 or the Type 1 complexes. The formation of these target capture complexes, which rapidly undergo the strand transfer chemistry, is the rate-limiting step in the overall reaction. These complexes provide alternate pathways to strand transfer, thereby maximizing transposition potential. This versatility is in contrast to other characterized transposons, which normally capture target DNA only at a single point in their respective reaction pathways.     

Path of DNA within the Mu transpososome. Transposase interactions bridging two Mu ends and the enhancer trap five DNA supercoils

The phage Mu transpososome is assembled by interactions of transposase subunits with the left (L) and right (R) ends of Mu and an enhancer (E) located in between. A metastable three-site complex LER progresses into a more stable type 0 complex in which a tetrameric transposase is poised for DNA cleavage. "Difference topology" has revealed five trapped negative supercoils within type 0, three contributed by crossings of E with L and R, and two by crossings of L with R. This is the most complex DNA arrangement seen to date within a recombination synapse. Contrary to the prevailing notion, the enhancer appears not to be released immediately following type 0 assembly. Difference topology provides a simple method for determining the ordered sequestration of DNA segments within nucleoprotein assemblies.     

Host DNA replication forks are not preferred targets for bacteriophage Mu transposition.

Bacteriophage Mu DNA integration in Escherichia coli strains infected after alignment of chromosomal replication was analyzed by a sandwich hybridization assay. The results indicated that Mu integrated into chromosomal segments at various distances from oriC with similar kinetics. In an extension of these studies, various Hfr strains were infected after alignment of chromosomal replication, and Mu transposition was shut down early after infection. The positions of integrated Mu copies were inferred from the transfer kinetics of Mu to an F- strain. Our analysis indicated that the location of Mu DNA in the host chromosome was not dependent on the positions of host replication forks at the time of infection. However, the procedure for aligning chromosomal replication affected DNA transfer by various Hfr strains differently, and this effect could account for prior results suggesting preferential integration of Mu at host replication forks.     

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.     

Transpososomes: stable protein-DNA complexes involved in the in vitro transposition of bacteriophage Mu DNA

We report that two types of stable protein-DNA complexes, or transpososomes, are generated in vitro during the Mu DNA strand transfer reaction. The Type 1 complex is an intermediate in the reaction. Its formation requires a supercoiled mini-Mu donor plasmid, Mu A and HU protein, and Mg2+. In the Type 1 complex the two ends of Mu are held together, creating a figure eight-shaped molecule with two independent topological domains; the Mu sequences remain supercoiled while the vector DNA is relaxed because of nicking. In the presence of Mu B protein, ATP, target DNA, and Mg2+, the Type 1 complex is converted into the protein-associated product of the strand transfer reaction. In this Type 2 complex, the target DNA has been joined to the Mu DNA ends held in the synaptic complex at the center of the figure eight. Supercoils are not required for the latter reaction.     

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.     

Role of DNA topology in Mu transposition: mechanism of sensing the relative orientation of two DNA segments

DNA strand transfer at the initiation of Mu transposition normally requires a negatively supercoiled transposon donor molecule, containing both ends of Mu in inverted repeat orientation. We propose that the specific relative orientation of the Mu ends is needed only to energetically favor a particular configuration that the ends must adopt in a synaptic complex. The model was tested by constructing special donor DNA substrates that, because of their catenation or knotting, energetically favor this same configuration of the Mu ends, even though they are on separate molecules or in direct repeat orientation. These structures are efficient substrates for the strand transfer reaction, whereas appropriate control structures are not. The result eliminates tracking or protein scaffold models for orientation preference. Several other systems in which the relative orientation of two DNA segments is sensed may utilize the same mechanism.     

Assembly of the active form of the transposase-Mu DNA complex: a critical control point in Mu transposition.

Discovery and characterization of a new intermediate in Mu DNA transposition allowed assembly of the transposition machinery to be separated from the chemical steps of recombination. This stable intermediate, which accumulates in the presence of Ca2+, consists of the two ends of the Mu DNA synapsed by a tetramer of the Mu transposase. Within this stable synaptic complex (SSC), the recombination sites are engaged but not yet cleaved. Thus, the SSC is structurally related to both the cleaved donor and strand transfer complexes, but precedes them on the transposition pathway. Once the active protein-DNA complex is constructed, it is conserved throughout transposition. The participation of internal sequence elements and accessory factors exclusively during SSC assembly allows recombination to be controlled prior to the irreversible chemical steps.     

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

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

Action at a distance in Mu DNA transposition: an enhancer-like element is the site of action of supercoiling relief activity by integration host factor (IHF).

The first committed step in the in vitro strand transfer reaction of a mini-Mu donor molecule is the formation of a Type 1 complex in which the Mu ends are held together in a non-covalent protein-DNA complex. Efficient formation of this complex at high levels of donor supercoiling (sigma approximately -0.06) requires the Mu A and Escherichia coli HU proteins. At in vivo levels of supercoiling, efficient reaction also requires E. coli integration host factor (IHF). We demonstrate that this supercoiling relief activity of IHF is mediated through an IHF binding site in the Mu early promoter region. This site is part of a larger enhancer-like element which includes operator 1 (01) and part of operator 2 (02) with the IHF site in between. The enhancer-like element stimulates the initial rate of the in vitro reaction 100-fold and acts in a distance-independent fashion. Inversion of the orientation of the element results in a total loss of enhancer activity in the absence of IHF. However, a 10-fold stimulation in the initial rate of reaction is induced by the addition of IHF. Furthermore, correct helical phasing between 01 and 02 is required for maximal activity. The results indicate that a specific geometrical configuration of the enhancer-like element, which includes a sharp bend between 01 and 02, is required for optimal induction of synapsis.     

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.     

Studies on a "jumping gene machine": higher-order nucleoprotein complexes in Mu DNA transposition.

Studies in my lab have focused on DNA transposition in the bacterial virus, Mu. In vitro studies have shown that Mu DNA transposition is a three-step process involving DNA breakage, strand transfer and DNA replication. In the first step, a nick is introduced at each end of the transposon. The liberated 3'-OH groups subsequently attack a target DNA molecule resulting in strand transfer. The transposon DNA, now covalently linked to the target, is finally replicated to generate the transposition end-product, referred to as a cointegrate. The DNA cleavage and strand transfer reactions are mediated by a "jumping gene machine" or transpososomes, which we discovered in 1987. They are assembled by bringing together three different DNA regions via a process involving multiple protein-DNA and protein-protein interactions. The action of four different proteins is required in addition to protein-induced DNA bending or wrapping to overcome the intrinsic stiffness of DNA, which would ordinarily prohibit the assembly of such a structure. Transpososome assembly is a gradual process involving multiple steps with an inherent flexibility whereby alternate pathways can be used in the assembly process, biasing the reaction towards completion under different conditions.     

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.     

The cis-acting DNA sequences required in vivo for bacteriophage Mu helper-mediated transposition and packaging

The 37,000 bp double-stranded DNA genome of bacteriophage Mu behaves as a plaque-forming transposable element of Escherichia coli. We have defined the cis-acting DNA sequences required in vivo for transposition and packaging of the viral genome by monitoring the transposition and maturation of Mu DNA-containing pSC101 and pBR322 plasmids with an induced helper Mu prophage to provide the trans-acting functions. We found that nucleotides 1 to 54 of the Mu left end define an essential domain for transposition, and that sequences between nucleotides 126 and 203, and between 203 and 1,699, define two auxiliary domains that stimulate transposition in vivo. At the right extremity, the essential sequences for transposition require not more than the first 62 base pairs (bp), although the presence of sequences between 63 and 117 bp from the right end increases the transposition frequency about 15-fold in our system. Finally, we have delineated the pac recognition site for DNA maturation to nucleotides 32 to 54 of the Mu left end which reside inside of the first transposase binding site (L1) located between nucleotides 1–30. Thus, the transposase binding site and packaging domains of bacteriophage Mu DNA can be separated into two well-defined regions which do not appear to overlap.Abbreviations attL attachment site left - attR attachment site right - bp base pairs - Kb kilobase pair - nt nucleotide - Pu Purine - Py pyrimidine - Tn transposable element State University of New York, Downstate Medical Center, Brooklyn, NY 11204 USA