Replication forks of Escherichia coli are not the preferred sites for lysogenic integration of bacteriophage Mu.

The question of whether bacteriophage Mu prefers replication forks for lysogenic integration into Escherichia coli chromosomes was tested by using two different systems. In the first, inactivation of genes was scored in synchronized cultures infected by Mu at various times. No increase in the mutation frequency of a gene was found after infection at the time of its replication. In the second, the composition of colonies formed by bacteria lysogenized by Mu was determined; the newly formed lysogens should give rise to mixed colonies (containing lysogenized as well as nonlysogenized bacteria), uniform colonies, or both, depending on the mode of integration. Both types of colonies were found, and the fraction of uniform colonies was proportional to the relative length of the unreplicated segment of an average chromosome in the culture. The results in both systems clearly preclude the possibility that a lysogenizing Mu integrates with high preference at the chromosome replication forks.     

Regulation of bacteriophage Mu transposition

Bacteriophage Mu is a transposon and a temperate phage which has become a paradigm for the study of the molecular mechanism of transposition. As a prophage, Mu has also been used to study some aspects of the influence of the host cell growth phase on the regulation of transposition. Through the years several host proteins have been identified which play a key role in the replication of the Mu genome by successive rounds of replicative transposition as well as in the maintenance of the repressed prophage state. In this review we have attempted to summarize all these findings with the purpose of emphasizing the benefit the virus and the host cell can gain from those phage-host interactions.     

A truncated form of the bacteriophage Mu B protein promotes conservative integration, but not replicative transposition, of Mu DNA

The phage-encoded proteins required for conservative integration of infecting bacteriophage Mu DNA were investigated. Our findings show that functional gpA, an essential component of the phage transposition system, is required for integration. The Mu B protein, which greatly enhances replicative transposition of Mu DNA, is also required. Furthermore, a truncated form of gpB lacking 18 amino acids from the carboxy terminus is blocked in replicative transposition, but not conservative integration. Our results point to a more prominent role for gpB than simply a replication enhancer in Mu DNA transposition. The ability of a truncated form of B to function in conservative integration, but not replicative transposition, also suggests a key role for the carboxy-terminal domain of the protein in the replicative reaction. The existence of a shortened form of gpB, which uncouples conservative integration from replicative transposition, should be invaluable for future dissection of Mu DNA transposition.     

Inhibition of bacteriophage Mu transposition by Mu repressor and Fis

In this paper we show that the Escherichia coli protein Fis has a regulatory function in Mu transposition in the presence of Mu repressor. Fis can lower the transposition frequency of a mini-Mu 3–80-fold, but only if the Mu repressor is expressed simultaneously. In this novel type of regulation of transposition by the concerted action of Fis and repressor, the IAS, the internal activating sequence, is also involved as deletion of this site leads to the loss of the Fis effect. As the IAS contains strong repressor binding sites these are probably the target for the repressor in the observed negative regulation by Fis and repressor. However, the role of Fis and repressor is not only to inactivate the IAS, since a 4bp insertion in the IAS, which changes the spacing of the repressor-binding site, abolishes the enhancing function of the IAS but leaves the repressor-Fis effect intact. A likely target for Fis in this regulation is a strong Fis-binding site, which is located adjacent to the L2 transposase-binding site. However, when this Fis-binding sequence was substituted by a random sequence and Fis no longer showed specific binding to this site, the Fis effect was still observed. Although it is still possible that Fis can function by binding to this non-specific site in a particular complex, it seems more likely that Fis is directly or indirectly involved in determining the level of the repressor.     

Electron microscopic analysis of in vitro transposition intermediates of bacteriophage Mu DNA

Bacteriophage Mu is a highly efficient transposon and the only moveable element for which an in vitro transposition system has been reported. Recently, this system has been used by Craigie and Mizuuchi [Cell 41 (1985) 867-876] to identify and biochemically characterize intermediates in the transposition process. We have utilized the in vitro transposition system to generate intermediates in the transposition process and have analyzed these intermediates by electron-microscopic methods. Partial denaturation mapping has shown the intermediates to be theta-shaped structures in which the phi X174 target DNA is joined to the mini-Mu plasmid at the ends of the Mu genome. Our results are in agreement with the previous biochemical studies and the type of intermediate we observe is exactly what is predicted by the Shapiro model of transposition [Proc. Natl. Acad. Sci. USA 76 (1979) 1933-1937].     

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     

Host cell DNA chain initiation protein requirements for replication of bacteriophage G4 replicative-form DNA.

Bacteriophages G4ev1 and G4bs1 are simple temperature-resistant derivatives of wild-type G4 as demonstrated by restriction endonuclease analyses. The rate of replication of the duplex replicative-form DNA of these phages was normal in dnaB and dnaC mutants of the host, whereas the rate was markedly reduced in a dnaG host mutant at the restrictive temperature. We conclude that G4 duplex DNA replication requires the host cell dnaG protein, but not the dnaB and dnaC proteins. The reasons for the differences between our conclusions and those based on previously published data are documented and discussed.     

DNA transposition of bacteriophage Mu. A quantitative analysis of target site selection in vitro.

The Mu transpositional DNA recombination machinery selects target sites by assembling a protein-DNA complex that interacts with the target DNA and reacts whenever it locates a favorable sequence composition. Splicing of a transposon into the target generates a 5-bp duplication that reflects the original target site. Preferential usage of different target pentamers was examined with a minimal Mu in vitro system and quantitatively compiled consensus sequences for the most preferred and the least preferred sites were generated. When analyzed as base steps, preferences toward certain steps along the 5-bp target site were detected. We further show that insertion sites can be predicted on the basis of additively calculated base step values. Also surrounding sequences influence the preference of a given pentamer; a symmetrical structural component was revealed, suggesting potential hinges at and around the target site.     

Mutants of Escherichia coli defective for replicative transposition of bacteriophage Mu.

We isolated 142 Hir- (host inhibition of replication) mutants of an Escherichia coli K-12 Mu cts Kil- lysogen that survived heat induction and the killing effect of Mu replicative transposition. All the 86 mutations induced by insertion of Tn5 or a kanamycin-resistant derivative of Tn10 and approximately one-third of the spontaneous mutations were found by P1 transduction to be linked to either zdh-201::Tn10 or Tn10-1230, indicating their location in or near himA or hip, respectively. For a representative group of these mutations, complementation by a plasmid carrying the himA+ gene or by a lambda hip+ transducing phage confirmed their identification as himA or hip mutations, respectively. Some of the remaining spontaneously occurring mutations were located in gyrA or gyrB, the genes encoding DNA gyrase. Mutations in gyrA were identified by P1 linkage to zei::Tn10 and a Nalr gyrA allele; those in gyrB were defined by linkage to tna::Tn10 and to a gyrB(Ts) allele. In strains carrying these gyrA or gyrB mutations, pBR322 plasmid DNA exhibited altered levels of supercoiling. The extent of growth of Mu cts differed in the various gyrase mutants tested. Phage production in one gyrA mutant was severely reduced, but it was only delayed and slightly reduced in other gyrA and gyrB mutants. In contrast, growth of a Kil- Mu was greatly reduced in all gyrase mutant hosts tested.     

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.     

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.     

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.     

Conditional coupling of leading-strand and lagging-strand DNA synthesis at bacteriophage T4 replication forks

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.     

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.     

UvsX recombinase and Dda helicase rescue stalled bacteriophage T4 DNA replication forks in vitro

The rescue of stalled replication forks via a series of steps that include fork regression, template switching, and fork restoration often has been proposed as a major mechanism for accurately bypassing non-coding DNA lesions. Bacteriophage T4 encodes almost all of the proteins required for its own DNA replication, recombination, and repair. Both recombination and recombination repair in T4 rely on UvsX, a RecA-like recombinase. We show here that UvsX plus the T4-encoded helicase Dda suffice to rescue stalled T4 replication forks in vitro. This rescue is based on two sequential template-switching reactions that allow DNA replication to bypass a non-coding DNA lesion in a non-mutagenic manner.     

Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks

In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a 'repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.     

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.     

Precise excision of bacteriophage Mu DNA

The temperate bacteriophage Mu is a transposable element that can integrate randomly into bacterial DNA, thereby creating mutations. Mutants due to an integrated Mu prophage do not give rise to revertants, as if Mu, unlike other transposable elements, were unable to excise precisely. In the present work, starting with a lacZ::Muc62(Ts) strain unable to form Lac+ colonies, we cloned a lacZ+ gene in vivo on a mini-Mu plasmid, under conditions of prophage induction. In all lac+ plasmids recovered, the wild-type sequence was restored in the region where the Mu prophage had been integrated. The recovery of lacZ+ genes shows that precise excision of Mu does indeed take place; the absence of Lac+ colonies suggests that precise excision events are systematically associated with loss of colony-forming ability.     

Structure-function relationships in the transposition protein B of bacteriophage Mu

The B-protein of phage Mu, which is required for high frequency intermolecular transposition in vivo, shows ATPase activity in vitro, binds nonspecifically to DNA, and stimulates intermolecular strand transfer. To elucidate the structural bases for B-protein function, it was subjected to limited proteolysis with two different proteases, trypsin and chymotrypsin. The resulting fragments were mapped by amino acid sequencing. These data show that the B-protein is organized in two domains: an amino-terminal domain of 25 kDa and a carboxyl-terminal domain of 8-kDa. A fragment analogous to the amino-terminal domain, produced by deleting the 3' end of a cloned B gene, proved to be insoluble and had to be renatured after elution from a sodium dodecyl sulfate gel. The renatured protein retains ATP-binding activity and to a lesser extent the DNA-binding activity of the MuB protein, but is unable to hydrolyze ATP or function in transposition. We also show in this study that efficient DNA-strand transfer by the B-protein occurs even in the absence of a detectable ATPase activity or in the presence of adenosine 5'-O-(thio)triphosphate (ATP gamma S).