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.

The wing of the enhancer-binding domain of Mu phage transposase is flexible and is essential for efficient transposition.

A tetramer of the Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Juxtaposition of the recombination sites is accomplished by the assembly of a stable synaptic complex of MuA protein and Mu DNA. This initial critical step is facilitated by the transient binding of the N-terminal domain of MuA to an enhancer DNA element within the Mu genome (called the internal activation sequence, IAS). Recently we solved the three-dimensional solution structure of the enhancer-binding domain of Mu phage transposase (residues 1-76, MuA76) and proposed a model for its interaction with the IAS element. Site-directed mutagenesis coupled with an in vitro transposition assay has been used to assess the validity of the model. We have identified five residues on the surface of MuA that are crucial for stable synaptic complex formation but dispensable for subsequent events in transposition. These mutations are located in the loop (wing) structure and recognition helix of the MuA76 domain of the transposase and do not seriously perturb the structure of the domain. Furthermore, in order to understand the dynamic behavior of the MuA76 domain prior to stable synaptic complex formation, we have measured heteronuclear 15N relaxation rates for the unbound MuA76 domain. In the DNA free state the backbone atoms of the helix-turn-helix motif are generally immobilized whereas the residues in the wing are highly flexible on the pico- to nanosecond time scale. Together these studies define the surface of MuA required for enhancement of transposition in vitro and suggest that a flexible loop in the MuA protein required for DNA recognition may become structurally ordered only upon DNA binding.

Assembly of phage Mu transpososomes: cooperative transitions assisted by protein and DNA scaffolds.

Transposition of phage Mu takes place within higher order protein-DNA complexes called transpososomes. These complexes contain the two Mu genome ends synapsed by a tetramer of Mu transposase (MuA). Transpososome assembly is tightly controlled by multiple protein and DNA sequence cofactors. We find that assembly can occur through two distinct pathways. One previously described pathway depends on an enhancer-like sequence element, the internal activation sequence (IAS). The second pathway depends on a MuB protein-target DNA complex. For both pathways, all four MuA monomers in the tetramer need to interact with an assembly-assisting element, either the IAS or MuB. However, once assembled, not all MuA monomers within the transpososome need to interact with MuB to capture MuB-bound target DNA. The multiple layers of control likely are used in vivo to ensure efficient rounds of DNA replication when needed, while minimizing unwanted transposition products.

Division of labor among monomers within the Mu transposase tetramer.

A single tetramer of Mu transposase (MuA) pairs the recombination sites, cleaves the donor DNA, and joins these ends to a target DNA by strand transfer. Analysis of C-terminal deletion derivatives of MuA reveals that a 30 amino acid region between residues 575 and 605 is critical for these three steps. Although inactive on its own, a deletion protein lacking this region assembles with the wild-type protein. These mixed tetramers carry out donor cleavage but do not promote strand transfer, even when the donor cleavage stage is bypassed. These data suggest that the active center of the transposase is composed of the C-terminus of four MuA monomers; one dimer carries out donor cleavage while all four monomers contribute to strand transfer.

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.

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.

MuB protein allosterically activates strand transfer by the transposase of phage Mu.

The MuA and MuB proteins collaborate to mediate efficient transposition of the phage Mu genome into many DNA target sites. MuA (the transposase) carries out all the DNA cleavage and joining steps. MuB stimulates strand transfer by activating the MuA-donor DNA complex through direct protein-protein contact. The C-terminal domain of MuA is required for this MuA-MuB interaction. Activation of strand transfer occurs irrespective of whether MuB is bound to target DNA. When high levels of MuA generate a pool of free MuB (not bound to DNA) or when chemical modification of MuB impairs its ability to bind DNA, MuB still stimulates strand transfer. However, under these conditions, intramolecular target sites are used exclusively because of their close proximity to the MuA-MuB-donor DNA complex.

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 sequence of the E. coli gyrB gene: application of a new sequencing strategy.

We have determined the sequence of the E. coli gyrB gene, using a new sequencing approach in which transposition from a mini-Mu plasmid into the DNA provides random start points for dideoxynucleotide sequence analysis. The gyrB sequence corresponds to a protein 804 amino acids long; a previously isolated protein fragment with partial enzymatic activity has been identified as the C-terminal half-molecule. A plausible terminator of gyrB transcription is located just beyond the structural gene.

DNA sequence of the control region of phage D108: the N-terminal amino acid sequences of repressor and transposase are similar both in phage D108 and in its relative, phage Mu.

We have determined the DNA sequence of the control region of phage D108 up to position 1419 at the left end of the phage genome. Open reading frames for the repressor gene, ner gene, and the 5' part of the A gene (which codes for transposase) are found in the sequence. The genetic organization of this region of phage D108 is quite similar to that of phage Mu in spite of considerable divergence, both in the nucleotide sequence and in the amino acid sequences of the regulatory proteins of the two phages. The N-terminal amino acid sequences of the transposases of the two phages also share only limited homology. On the other hand, a significant amino acid sequence homology was found within each phage between the N-terminal parts of the repressor and transposase. We propose that the N-terminal domains of the repressor and transposase of each phage interact functionally in the process of making the decision between the lytic and the lysogenic mode of growth.

The extent of DNA sequence required for a functional bacterial attachment site of phage lambda.

We have investigated the extent of DNA sequence required to form a bacterial attachment site (attB) that functions in bacteriophage lambda integration. A DNA fragment carrying attB of Escherichia coli was trimmed, recloned and tested for recombination proficiency. We found that the common core sequence plus the adjoining 4-bp sequences of both the B and B' arms are required for full activity, while plasmids with an even shorter attB sequence retain some capacity to function as attB in vivo. We also found that the nonspecific DNA that is joined to the required attachment site sequence does not significantly influence the rate of the recombination reaction.

Site-specific recognition of the bacteriophage Mu ends by the Mu A protein.

The Mu A protein binds site-specifically to the ends of Mu DNA. Two blocks of protection against nuclease are seen at the left (L) end; the right (R) end exhibits one continuous block of protection. We interpret the nuclease protection pattern and sequence data as evidence for three Mu A protein binding sites at each end of Mu. Both the L and R ends have one site close to the terminus; each end also has two additional sites that differ in location between the L and R ends. The Mu A protein protection patterns on the L ends of Mu and the closely related phage D108 are, despite many interspersed sequence differences in one of the protected regions, essentially identical. We show that the A proteins of Mu and D108 can function, at different efficiencies, interchangeably on the Mu and D108 L ends in vivo. Purified Mu repressor, in addition to its primary binding in the operator region, also binds less strongly to the Mu ends at the same sites as the Mu A protein. This affinity of Mu repressor for DNA sites recognized by the Mu A protein may play a role as a second level of control of transposition by the repressor.

Cloning and simplified purification of Escherichia coli DNA gyrase A and B proteins.

We have transferred the Escherichia coli gyrA and gyrB genes onto plasmids that allow the overproduction of the DNA gyrase A and B proteins and have designed relatively simple purification procedures for both proteins. The pure proteins are obtained in good yield; from 2 liters of culture (12 g of cells), one can recover 25 mg of GyrA or 3 mg of GyrB protein.

Integrative recombination of bacteriophage lambda: extent of the DNA sequence involved in attachment site function.

We have investigated the minimum extent of DNA sequence required for the attachment site of bacteriophage lambda to function in integrative recombination. A DNA fragment carrying the phage attachment site (attP) of bacteriophage lambda was trimmed, recloned, and tested for recombination proficiency. In order to recombine with the bacterial attachment site (attB), the phage attachment site must retain about 250 base pairs of its original sequence. On the left side, the essential sequence extends beyond 106 base pairs from the center of the 15-base-pair common core sequence but not beyond 152 base pairs. On the right side the required sequence extends beyond 68 base pairs but not beyond 99 base pairs from the center of the core. A trimmed site that has lost part of the sequence mentioned above cannot function as the phage attachment site. However, depending on which part of the sequence is present, such a site can still act in reactions normally requiring one of the prophage attachment sites or the bacterial attachment site. The results also suggest that the essential suquence of the bacterial attachment site consists only of the sequence common to the phage and bacterial attachment sites.