Bacteriophage Mu DNA replication in vitro

An in vitro system for bacteriophage Mu DNA replication using lysates on cellophane discs is described. Mu replication was monitored by DNA hybridization. Using a thermoinducible Mu lysogen, 30-50% of all DNA synthesis in vitro was Mu-specific. Mu DNA synthesis is semidiscontinuous. In the presence of the DNA ligase inhibitor NMN, about one-half of the DNA was in Okazaki pieces and one-half in large DNA. The Mu Okazaki pieces hybridized mainly to the Mu light strand; the large DNA hybridized mainly to the Mu heavy strand. Okazaki pieces isolated from uninfected cells also hybridized to 2000-3000 bases of host DNA present in Mu-separated strands. However, the host Okazaki pieces hybridize to both Mu strands symmetrically. Most, if not all, host sequences were represented in mature Mu viral DNA. The in vitro data are most consistent with models in which Mu sequences, oriented randomly in both directions in the host chromosome, have recruited a bacterial replisome which traverses the Mu genome from left to right.     

Mu DNA replication in vitro: Criteria for initiation

Summary An in vitro system for investigating Mu replication and transposition using film lysates has recently been described (Higgins et al. 1983). Under most conditions examined, little or no replication initiation takes place in vitro. The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro. Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates.Two conditions reported to specifically decrease Mu DNA replication in vivo (Pato and Reich 1982) were assessed for their effects on in vitro replication. Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates. However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro. These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates. The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro.     

Early events in the replication of Mu prophage DNA.

To determine whether the early replication of Mu prophage DNA proceeds beyond the termini of the prophage into hose DNA, the amounts of both Mu DNA and the prophage-adjacent host DNA sequences were measured using a DNA-DNA annealing assay after induction of the Mu vegetative cycle. Whereas Mu-specific DNA synthesis began 6 to 8 min after induction, no amplification of the adjacent DNA sequences was observed. These data suggest that early Mu-induced DNA synthesis is constrained within the boundaries of the Mu prophage. Since prophage Mu DNA does not undergo a prophage lambda-like excision from its original site after induction (E. Ljungquist and A. I. Bukhari, Proc. Natl. Acad. Sci. U.S.A. 74:3143--3147, 1977), we propose the existence of a control mechanism which excludes prophage-adjacent sequences from the initial mu prophage replication. The frequencies of the Mu prophage-adjacent DNA sequences, relative to other Escherichia coli genes, were not observed to change after the onset of Mu-specific DNA replication. This suggests that these regions remain associated with the host chromosome and continue to be replicated by the chromosomal replication fork. Therefore, we conclude that both the Mu prophage and adjacent host sequences are maintained in the host chromosome, rather than on an extrachromosomal form containing Mu and host DNA.     

The phiX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition.

Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the phiX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate.     

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.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Instability of transposase activity: Evidence from bacteriophage Mu DNA replication

Transposition of genetic elements involves coupled replication and integration events catalyzed in part by a class of proteins called transposases. We have asked whether the transposase activity of bacteriophage Mu (the Mu A protein) is stable and capable of catalyzing multiple rounds of coupled replication/integration, or whether its continued synthesis is required to maintain Mu DNA replication. Inhibition of protein synthesis during the lytic cycle with chloramphenicol inhibited Mu DNA synthesis with a half-life of approximately 3 min, demonstrating a need for continued protein synthesis to maintain Mu DNA replication. Synthesis of specific Mu-encoded proteins was inhibited by infecting a host carrying a temperature-sensitive suppressor, at permissive temperature, with Mu amber phages, then shifting to nonpermissive temperature. When Aam phages were used, Mu DNA replication was inhibited with kinetics essentially identical to those with chloramphenicol addition; hence, it is likely that continued synthesis of the Mu A protein is required to maintain Mu DNA replication. The data suggest that the activity of the Mu A protein is unstable, and raise the possibility that the Mu A protein and other transposases may be used stoichiometrically rather than catalytically.     

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.     

Stoichiometric use of the transposase of bacteriophage Mu

The transposase of bacteriophage Mu (gene A protein) mediates the coupled replication and integration processes that constitute transposition during the lytic cycle. Our previous results showed that the activity of the A protein is unstable, as its continued synthesis is required to maintain Mu DNA replication throughout the lytic cycle. We present here the results of experiments in which the A protein is used stoichiometrically and must be synthesized de novo for each round of Mu DNA replication. Induction of a Mu lysogen in the absence of DNA replication allows accumulation of potential for a single round of Mu DNA replication. Once achieved, this potential is stable even in the absence of further protein synthesis. Release of inhibition of DNA replication leads to a single semi-conservative replicative transposition event, followed by later rounds only if additional synthesis of the A protein is allowed.     

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.     

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.     

Synchronization of bacteriophage Mu DNA replicative transposition: analysis of the first round after induction.

The lytic cycle of bacteriophage Mu includes a large number of coupled DNA replication and integration events, each of which is equivalent in several respects to the process of transposition of genetic elements. To aid us in studying the process of Mu DNA replicative transposition, we developed a technique for synchronizing the first round of replication following induction of a lysogen. Synchronization was achieved by inducing a lysogen in the absence of DNA replication for a time sufficient to develop the potential for Mu DNA replication in all cells in the population; upon release of the inhibition of replication, a synchronized round of Mu DNA replication was observed. Development of the potential for Mu DNA replication in the entire population took approximately 12 min. Protein synthesis was required for development of the potential, but the requirement for protein synthesis was satisfied by approximately 9 min suggesting that other, as yet unspecified, reactions occupied the last 3 min. Replication proceeded predominantly from the left end of the prophage, though a significant amount of initiation from the right end was observed. The usefulness of the technique for studying the mechanism of replicative transposition and the end products of a single round of replication are discussed.     

Chloroplast and Nuclear DNA Content of Cultured Spinach Leaf Discs

The amounts of chloroplast DNA and nuclear DNA in the cellsof spinach leaf discs cultutred under different light regimeshave been measured. The cellular level of ctDNA increased 10-foldin discs cultured in white light and was accompanied by a 2-foldincrease in the cellular level of nuclear DNA. In low intensitygreen light the cellular level of ctDNA increased 6-fold butwas not accompanied by an increase in the level of nuclear DNA.No net DNA synthesis on a per cell basis occurred in discs culturedin darkness. Chloroplasts of uncultured leaf discs containedan average of 83 plastome copies. The number of plastome copiesper chloroplast after 6 days culture decreased to 36 copiesin darkness, remained almost constant at 73 copies in whitelight and increased to 215 copies in low intensity green light. These results suggest that ctDNA replication can be independentof cellular levels of nuclear DNA and chloroplast division.     

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.     

T antigen and template requirements for SV40 DNA replication in vitro.

A cell-free system for replication of SV40 DNA was used to assess the effect of mutations altering either the SV40 origin of DNA replication or the virus-encoded large tumor (T) antigen. Plasmid DNAs containing various portions of the SV40 genome that surround the origin of DNA replication support efficient DNA synthesis in vitro and in vivo. Deletion of DNA sequences adjacent to the binding sites for T antigen either reduce or prevent DNA synthesis. This analysis shows that sequences that had been previously defined by studies in vivo to constitute the minimal core origin sequences are also necessary for DNA synthesis in vitro. Five mutant T antigens containing amino acid substitutions that affect SV40 replication have been purified and their in vitro properties compared with the purified wild-type protein. One protein is completely defective in the ATPase activity of T antigen, but still binds to the origin sequences. Three altered proteins are defective in their ability to bind to origin DNA, but retain ATPase activity. Finally, one of the altered T antigens binds to origin sequences and contains ATPase activity and thus appears like wild-type for these functions. All five proteins fail to support SV40 DNA replication in vitro. Interestingly, in mixing experiments, all five proteins efficiently compete with the wild-type protein and reduce the amount of DNA replication. These data suggest that an additional function of T antigen other than origin binding or ATPase activity, is required for initiation of DNA replication.     

Behavior of bacteriophage Mu DNA upon infecton of Escherichia coli cells.

The question whether the ends of bacteriophage Mu DNA are fused to form a ring in host cells is critical to the understanding of the mechanism of integrative recombination between Mu DNA and host DNA. We have examined the fate of ³²P-labeled Mu DNA, after infection of sensitive and immune (lysogenic) cells, by sedimentation in sucrose gradients, ethidium bromide/CsCl density centrifugation and by electrophoresis of parental Mu DNA and its fragments in agarose gels. We find that the parental Mu DNA cannot be detected as covalently closed circles at any stage during the Mu life cycle. An interesting form of Mu DNA can be seen after superinfection of immune cells. This form sediments about twice as fast as the mature phage DNA marker in neutral sucrose gradients but yields linear molecules upon phenol extraction. Upon infection of sensitive cells, most of the parental DNA associates with a large complex, presumably containing the host chromosome. When Mu-sensitive cells are infected with unlabeled Mu particles and Mu DNA examined at different times after infection by fractionation in 0.3% agarose gels and hybridization with ³²P-labeled Mu DNA, Mu sequences are found to appear with the bulk host DNA as the phage lytic cycle progresses. However, no distinct replicative or integrative intermediate of Mu, that behaves differently from linear Mu DNA and is separate from the host DNA, can be detected.     

Neighboring plasmid sequences can affect Mini-Mu DNA transposition in the absence of expression of the bacteriophage Mu semi-essential early region

Bacteriophage Mu DNA, like other transposable elements, requires DNA sequences at both extremities to transpose. It has been previously demonstrated that the transposition activity of various transposons can be influenced by sequences outside their ends. We have found that alterations in the neighboring plasmid sequences near the right extremity of a Mini-Mu, inserted in the plasmid pSC101, can exert an influence on the efficiency of Mini-Mu DNA transposition when an induced helper Mu prophage contains a polar insertion in its semi-essential early region (SEER). The SEER of Mu is known to contain several genes that can affect DNA transposition, and our results suggest that some function(s), located in the SEER of Mu, may be required for optimizing transposition (and thus, replication) of Mu genomes from restrictive locations during the lytic cycle.     

Kinetics of Mu DNA synthesis

Summary Mu specific DNA synthesis starts at 10 min after infection. All essential amber mutants of Mu were tested for the ability to replicate in a non permissive host. Except for the amber mutants A and B, which were already known to be blocked in Mu DNA synthesis (Wijffelman et al., 1974), all the other mutants showed normal Mu DNA replication.Using mitomycin C-treated cells Mu DNA synthesis was found to start at about 20 min after induction. However using the much more sensitive method of DNA-RNA hybridization, it was found that the DNA synthesis starts already at 10 min after induction, and that at 20 min after induction about 7 copies of the Mu DNA are present per cell.     

Dissection of the bacteriophage Mu strong gyrase site (SGS): significance of the SGS right arm in Mu biology and DNA gyrase mechanism

The bacteriophage Mu strong gyrase site (SGS), required for efficient phage DNA replication, differs from other gyrase sites in the efficiency of gyrase binding coupled with a highly processive supercoiling activity. Genetic studies have implicated the right arm of the SGS as a key structural feature for promoting rapid Mu replication. Here, we show that deletion of the distal portion of the right arm abolishes efficient binding, cleavage, and supercoiling by DNA gyrase in vitro. DNase I footprinting analysis of the intact SGS revealed an adenylyl imidodiphosphate-dependent change in protection in the right arm, indicating that this arm likely forms the T segment that is passed through the cleaved G segment during the supercoiling reaction. Furthermore, in an SGS derivative with an altered right-arm sequence, the left arm showed these changes, suggesting that the selection of a T segment by gyrase is determined primarily by the sequences of the arms. Analysis of the sequences of the SGS and other gyrase sites suggests that the choice of T segment correlates with which arm possesses the more extensive set of phased anisotropic bending signals, with the Mu right arm possessing an unusually extended set of such signals. The implications of these observations for the structure of the gyrase-DNA complex and for the biological function of the Mu SGS are discussed.