Immunoelectron microscopic analysis of the A, B, and HU protein content of bacteriophage Mu transpososomes

Stable protein-DNA complexes or transpososomes mediate the Mu DNA strand transfer reaction in vitro (Surette, M. G., Buch, S. J., and Chaconas, G. (1987) Cell 49, 253-262; Craigie, R., and Mizuuchi, K. (1987) Cell 51, 493-501). Formation of the Type 1 complex, an intermediate in the strand transfer reaction, requires the Mu A and Escherichia coli HU proteins. Generation of the Type 2 complex, in which the Mu ends have been covalently linked to the target DNA, requires the Mu B protein, ATP, and target DNA in addition to A and HU. The protein content of these higher order synaptic complexes has been studied by immunoelectron microscopy using protein A-colloidal gold conjugates to visualize antibody-bound complexes. Under our in vitro transposition conditions, Type 1 complexes were found to contain A and HU; in addition, Type 2 complexes contained Mu B. However, both the HU and the Mu B protein were found to be loosely associated and could be quantitatively removed from the nucleoprotein core of both complexes by incubation in 0.5 M NaCl. Depletion of HU from the Type 1 complex did not affect the ability of this complex to be converted into the strand-transferred product. Hence, the indispensable role of the HU protein in the Mu DNA strand transfer reaction is limited to the formation of the Type 1 transpososome.     

Transposition of Mu DNA: joining of Mu to target DNA can be uncoupled from cleavage at the ends of Mu

Transposition of Mu involves transfer of the 3' ends of Mu DNA to the 5' ends of a staggered cut in the target DNA. We find that cleavage at the 3' ends of Mu DNA precedes cutting of the target DNA. The resulting nicked species exists as a noncovalent nucleoprotein complex in which the two Mu ends are held together. This cleaved donor complex completes strand transfer when a target DNA, Mu B protein, and ATP are provided. Mu end DNA sequences that have been precisely cut at their 3' ends by a restriction endonuclease, instead of by Mu A protein and HU, are efficiently transferred to a target DNA upon subsequent incubation with Mu A protein, Mu B protein, and ATP. Cleavage of the Mu ends therefore cannot be energetically coupled with joining these ends to a target DNA. We discuss the DNA strand transfer mechanism in view of these results, and propose a model involving direct transfer of the 5' ends of the cut target DNA, from their original partners, to the 3' ends of Mu.     

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.     

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.     

Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome.

The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] foot-printing, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.     

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.     

Flanking host sequences can exert an inhibitory effect on the cleavage step of the in vitro mu DNA strand transfer reaction.

The effect of flanking host sequences on the cleavage step of the in vitro Mu DNA strand transfer reaction was investigated. Insertion of a mini-Mu molecule into certain sites in pUC19 results in insertions that demonstrate a decreased ability to form Type 1 complexes in subsequent rounds of transposition. Similarly, changes in the flanking host sequences directly adjacent to the Mu ends by in vitro mutagenesis can also result in Type 1-deficient mini-Mu molecules. Further examination of the inhibition revealed that Type 1 deficient mini-Mu molecules are capable of forming uncut synaptic complexes at normal levels but are compromised in their ability to serve as substrates for phosphodiester bond hydrolysis at the Mu ends. This cleavage defect can be overcome by addition of the Mu B protein and ATP to the reaction. Our data suggest that one of the roles of the B protein may be to provide a mechanism whereby Mu prophages with inhibitory flanking sequences can overcome this obstacle and avoid being trapped at unproductive locations.     

Inversion of the phosphate chirality at the target site of Mu DNA strand transfer: evidence for a one-step transesterification mechanism.

Central to transposition of phage Mu are two reactions mediated by the MuA protein. First, MuA introduces single-stranded cuts at the ends of the Mu DNA to generate 3' OH termini. In the subsequent strand-transfer step, the MuA-Mu DNA end complex cuts a target DNA and joins the Mu 3' ends to the 5' ends of the target. DNA containing chiral phosphorothioates was used to demonstrate inversion of the chirality during the course of strand transfer. This result strongly supports a one-step transesterification mechanism in which the 3' OH of the cleaved donor DNA is the attacking nucleophile. Furthermore, this donor 3' OH group was essential for target DNA cleavage. In contrast, during lambda integration the phosphate chirality was retained, as expected for a two-step transesterification involving a covalent protein-DNA intermediate.     

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.     

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.     

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.     

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

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

Mechanism of Mu DNA transposition

The study of Mu DNA transposition in vitro has resulted in a much better understanding of the biochemical details of the transposition process. An early step in transposition is the generation of a 5th structure which is the product of the strand-tansfer reaction. The polarity of the strand transfer has been determined and substantial progress has been made on the role of the individual proteins. Moreover, the strand-transfer reaction is mediated by stable protein–DNA complexes, or transposomes, and the reaction can be divided into two sequential steps. The role of the transposomes and the requirement for a supercoiled Mu DNA substrate are also discussed.     

Stimulation of the Mu DNA strand cleavage and intramolecular strand transfer reactions by the Mu B protein is independent of stable binding of the Mu B protein to DNA.

Interactions between the Mu A and Mu B proteins are important in the early steps of the in vitro transposition of a mini-Mu plasmid. We have examined these interactions by assaying Mu B stimulation of Mu A-mediated strand cleavage and strand transfer reactions. We have previously shown that in the presence of ATP the Mu B protein can stimulate the Mu A-directed cleavage reaction of mini-Mu plasmids carrying a terminal base pair mutation (Surette, M.G., Harkness, T., and Chaconas, G. (1991) J. Biol. Chem. 266, 3118-3124). Here we demonstrate that in the absence of a non-Mu DNA target molecule the Mu B protein stimulates intramolecular integration of a mini-Mu in an ATP-dependent fashion. Furthermore, modification of the Mu B protein with N-ethylmaleimide severely compromises the ability of B to form a stable complex with DNA; however, the modified protein stimulates the strand cleavage and intramolecular strand transfer reactions as efficiently as the untreated protein. These results indicate that the Mu B protein is capable of stimulating the Mu A protein through direct interaction in the absence of stable Mu B-DNA complex formation. Our results increase the spectrum of Mu B protein activities and uncouple the stimulatory properties of the Mu B protein from stable DNA binding but not the ATP cofactor requirement.     

Role of the A protein-binding sites in the in vitro transposition of mu DNA. A complex circuit of interactions involving the mu ends and the transpositional enhancer.

To investigate the role of the A protein-binding sites at the Mu ends in the DNA strand transfer reaction, we constructed mutant mini-Mu molecules in which these sites were deleted (L3 or R3) or substituted (L2 or R2) to conserve the spacing arrangements at the adjacent sites. The single site mutants are poor substrates for phosphodiester bond hydrolysis at the Mu ends in Type 1 reactions in the absence of Escherichia coli integration host factor (IHF). Addition of IHF to the reaction stimulates Type 1 cleavage more than 10 times for the delta-R3, delta-L3, S-L2 mutants and more than five times in the case of the S-R2 mutant under alternate conditions. The site of IHF stimulation resides within the transpositional enhancer which implicates the end-binding sites L2, L3, R2, and R3 in interactions with the enhancer. At least two of the L2, L3, and R3 sites are required for proficient reaction in the presence of IHF. By combining the single site mutants with O1 or O2 partially deleted enhancer elements, we have tentatively localized some of the interactions to each side of the functional enhancer revealing a complex circuit of end-enhancer interactions. The R3 site is suggested to be involved in interactions only with O2 and the L3 site only with O1. The data also suggest the possibility that L2 and R2 may be involved in interactions with both O1 and O2. Finally, our working model predicts that the L3-O1 and R3-O2 interactions may be required contacts for discriminating between the Mu left and right ends in transpososome formation.     

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.     

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.     

Two mutations of phage mu transposase that affect strand transfer or interactions with B protein lie in distinct polypeptide domains.

Two mutations within the transposase (the A protein) gene of phage Mu with distinct effects on DNA transposition have been studied. The first mutation maps to the central domain (domain II) of A, a protein consisting of three major structural domains. The variant protein is normal in synapsis and cleavage of Mu ends but is temperature-sensitive in the strand transfer reaction, joining the Mu ends to target DNA. The second mutation is a deletion at the C terminus (within domain III); on the basis of genetic studies, the mutant protein is predicted to have lost the ability to interact with the Mu B protein. The B protein, in conjunction with A, promotes efficient intermolecular transposition, while inhibiting intramolecular transposition. We show that the purified mutant protein is proficient in intramolecular, but not intermolecular transposition in vitro. The interactions between A and B proteins have been followed by a proteolysis assay. The chymotrypsin sensitivity of the interdomainal Phe221-Ser222 peptide bond within the bidomainally organized B protein is exquisitely modulated by ATP, DNA and A protein. The sensitive or "open" state of this bond in native B protein becomes partially "open" upon binding of ATP by B, attains a "closed" or resistant configuration upon binding of DNA in presence of ATP, and is rendered "open" again upon addition of the A protein. In this test for the interaction of A protein with B protein-DNA complex, the domain II mutant behaves like wild-type A protein. However, the domain III mutant fails to restore chymotrypsin susceptibility of the Phe221-Ser222 bond.     

Human immunodeficiency virus type 1 nucleocapsid protein specifically stimulates Mg2+-dependent DNA integration in vitro.

The integrase (IN) protein of the human immunodeficiency virus mediates integration of the viral DNA into the cellular genome. In vitro, this reaction can be mimicked by using purified recombinant IN and model DNA substrates. IN mediates two reactions: an endonucleolytic cleavage at each 3' end of the proviral DNA (terminal cleavage) and the joining of the linear viral DNA to 5' phosphates in the target DNA (strand transfer). Previous investigators have shown that purified IN requires Mn2+ or Mg2+ to promote strand transfer in vitro, although Mg2+ is the likely metal cofactor in vivo. IN activity in the presence of Mg2+ in vitro requires high IN concentrations and low concentrations of salt. Here, we show that the viral nucleocapsid protein NCp7 allows efficient IN-mediated strand transfer in the presence of Mg2+ at low enzyme concentrations. This potentiating effect appears to be unique to NCp7, as other small DNA-binding proteins, while capable of stimulating integration in the presence of Mn2+, all failed to stimulate strand transfer in the presence of Mg2+.     

Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition.

Tn10 transposes by a nonreplicative "cut and paste" mechanism. We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site. The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction. These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition. These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes. The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.