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.     

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

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

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd2(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.     

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.     

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.     

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

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

DNA intermediates in transposition of phage Mu

Transposable genetic elements can insert into DNA sites that have no homology to themselves. Evidence that there is a physical linkage between a transposable element and its target DNA sequence during transposition comes from studies on bacteriophage Mu DNA transposition in which plasmids containing Mu DNA have been shown to attach to host DNA. We report the isolation of key structures, seen after induction of Mu DNA replication, after cloning lac operator into Mu DNA and using the lac repressor-operator interaction to trap Mu DNA on nitrocellulose filters. We have localized Mu sequences within these structures in the electron microscope by visualizing the lac operator-repressor interaction after binding with ferritin-conjugated antibody. This analysis shows that key structures contain replicating Mu DNA linked to non-Mu DNA and that replication can begin at either end of Mu.     

Solution conformations of early intermediates in Mos1 transposition

DNA transposases facilitate genome rearrangements by moving DNA transposons around and between genomes by a cut-and-paste mechanism. DNA transposition proceeds in an ordered series of nucleoprotein complexes that coordinate pairing and cleavage of the transposon ends and integration of the cleaved ends at a new genomic site. Transposition is initiated by transposase recognition of the inverted repeat sequences marking each transposon end. Using a combination of solution scattering and biochemical techniques, we have determined the solution conformations and stoichiometries of DNA-free Mos1 transposase and of the transposase bound to a single transposon end. We show that Mos1 transposase is an elongated homodimer in the absence of DNA and that the N-terminal 55 residues, containing the first helix-turn-helix motif, are required for dimerization. This arrangement is remarkably different from the compact, crossed architecture of the dimer in the Mos1 paired-end complex (PEC). The transposase remains elongated when bound to a single-transposon end in a pre-cleavage complex, and the DNA is bound predominantly to one transposase monomer. We propose that a conformational change in the single-end complex, involving rotation of one half of the transposase along with binding of a second transposon end, could facilitate PEC assembly.     

Factors affecting transposition of the Himar1 mariner transposon in vitro.

Mariner family transposable elements are widespread in animals, but their regulation is poorly understood, partly because only two are known to be functional. These are particular copies of the Dmmar1 element from Drosophila mauritiana, for example, Mos1, and the consensus sequence of the Himar1 element from the horn fly, Haematobia irritans. An in vitro transposition system was refined to investigate several parameters that influence the transposition of Himar1. Transposition products accumulated linearly over a period of 6 hr. Transposition frequency increased with temperature and was dependent on Mg2+ concentration. Transposition frequency peaked over a narrow range of transposase concentration. The decline at higher concentrations, a phenomenon observed in vivo with Mos1, supports the suggestion that mariners may be regulated in part by "overproduction inhibition." Transposition frequency decreased exponentially with increasing transposon size and was affected by the sequence of the flanking DNA of the donor site. A noticeable bias in target site usage suggests a preference for insertion into bent or bendable DNA sequences rather than any specific nucleotide sequences beyond the TA target site.     

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     

Characterization of functionally important sites in the bacteriophage Mu transposase protein

The 663 amino acid Mu transposase protein is absolutely required for Mu DNA transposition. Mutant proteins were constructed in vitro in order to locate regions of transposase that may be important for the catalysis of DNA transposition. Deletions in the A gene, which encodes the transposase, yielded two stable mutant proteins that aid in defining the end-specific DNA-binding domain. Linker insertion mutagenesis at eight sites in the Mu A gene generated two proteins, FF6 and FF14 (resulting from two and four amino acid insertions, respectively, at position 408), which were thermolabile for DNA binding in vitro at 43°C. However, transposition activity in vivo was severely reduced for all mutant proteins at 37°C, except those with insertions at positions 328 and 624. In addition, site-specific mutagenesis was performed to alter tyrosine 414, which is situated in a region that displays amino acid homology to the active sites of a number of nicking/closing enzymes. Tyrosine 414 may reside within an important, yet non-essential, site of transposase, as an aspartate-substituted protein had a drastically reduced frequency of transposition, while the remaining mutants yielded reduced, but substantial, frequencies of Mu transposition in vivo.     

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.     

A novel DNA binding and nuclease activity in domain III of Mu transposase: evidence for a catalytic region involved in donor cleavage.

The Mu A protein is a 75 kDa transposase organized into three structural domains. By severing the C-terminal region (domain III) from the remainder of the protein, we unmasked a novel non-specific DNA binding and nuclease activity in this region. Deletion analysis localized both activities to a 26 amino acid stretch (aa 575-600) which remarkably remained active in DNA binding and cleavage. The two activities were shown to be tightly linked by site-directed mutagenesis. To study the importance of these activities in the transposition process, an intact mutant transposase lacking the DNA binding and nuclease activity of domain III was constructed and purified. The mutant transposase was indistinguishable from wild-type Mu A in binding affinity for both the Mu ends and the enhancer, and in strand transfer activity when the cleavage step was bypassed. In contrast, the mutant transposase displayed defects in both synapsis and donor cleavage. Our results strongly suggest that the 26 amino acid region in domain III carries catalytic residues required for donor DNA cleavage by Mu A protein. Furthermore, our data suggest that an active site for donor cleavage activity in the Mu tetramer is assembled from domain II (metal ion binding) in one A monomer and domain III (DNA cleavage) in a separate A monomer. This proposal for active site assembly is in agreement with the recently proposed domain sharing model by Yang et al. (Yang, J.Y., Kim, K., Jayaram, M. and Harshey, R.M. [1995] EMBO J., 14, 2374-2384).     

The organization of the outside end of transposon Tn5.

The end sequences of the IS50 insertion sequence are known as the outside end (OE) and inside end. These complex ends are related but nonidentical 19-bp sequences that serve as substrates for the activity of the Tn5 transposase. Besides providing the binding site of the transposase, the end sequences of a transposon contain additional types of information necessary for transposition. These additional properties include but are not limited to host protein interaction sites and sites that program synapsis and cleavage events. In order to delineate the properties of the IS50 ends,the base pairs involved in the transposase binding site have been defined. This has been approached through performing a variety of in vitro analyses: a ++hydroxyl radical missing-nucleoside interference experiment, a dimethyl sulfate interference experiment, and an examination of the relative binding affinities of single-site end substitutions. These approaches have led to the conclusion that the transposase binds to two nonsymmetrical regions of the OE, including positions 6 to 9 and 13 to 19. Proper binding occurs along one face of the helix, over two major and minor grooves, and appears to result in a significant bending of the DNA centered approximately 3 bp from the donor DNA-OE junction.     

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.     

P element transposition in vitro proceeds by a cut-and-paste mechanism and uses GTP as a cofactor.

We have developed an in vitro reaction system for Drosophila P element transposition. Transposition products were recovered by selection in E. coli, and contained simple P element insertions flanked by 8 bp target site duplications as observed in vivo. Transposition required Mg+2 and partially purified P element transposase. Unlike other DNA rearrangement reactions, P element transposition in vitro used GTP as a cofactor; deoxyGTP, dideoxyGTP, or the nonhydrolyzable GTP analogs GMP-PNP or GMP-PCP were also used. Transposon DNA molecules cleaved at the P element termini were able to transpose, but those lacking 3'-hydroxyl groups were inactive. These biochemical data are consistent with genetic data suggesting that P element transposition occurs via a "cut-and-paste" mechanism.     

Interactions of the transposase with the ends of Mu: formation of specific nucleoprotein structures and non-cooperative binding of the transposase to its binding sites.

Transposition of the E. coli bacteriophage Mu requires the phage encoded A and B proteins, the host protein HU and the host replication proteins. The ends of the genome of the phage, on which some of these proteins act, both contain three transposase (A) binding sites. The organization of these binding sites on each end, however, is different. Here we show, using DNase footprinting experiments with purified A protein, that mutant A binding sites, which affect transposition, have decreased affinity for the transposase. Furthermore the transposase binds non-cooperatively to all A binding sites both in the left and right end of Mu. Electron microscopic studies show that the A protein forms specific nucleoprotein structures upon binding to the ends of Mu. The A and B proteins interact with the ends of Mu to generate larger structures than with the A protein alone.     

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

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

Transposition without transposase: a spontaneous mutation in bacteria.

Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.