Infecting bacteriophage mu DNA forms a circular DNA-protein complex

Upon superinfection of immune (lysogenic) cells with bacteriophage Mu, a form of Mu DNA accumulates that sediments about twice as fast as the linear phage DNA marker in neutral sucrose gradients. This form is also detected upon infection of sensitive cells with Mu. We have purified it and examined its physical nature. Under the electron microscope it appears circular and supertwisted. Upon treatment with Pronase, phenol or sodium dodecyl sulfate, however, it is converted to a linear Mu-length form, indicating that the circle is not covalently closed. The linear DNA still has heterogeneous host sequences at its termini. The circular DNA is resistant to the action of Escherichia coli exonuclease III and T7 exonuclease, but becomes sensitive to these nucleases after treatment with Pronase showing the presence of a protein that binds non-covalently to the ends of the DNA to circularize it as well as protect it from digestion with exonucleases. The complex is resistant to high salt (up to 6 M-NaCl) but can undergo transitions between forms that are partially open, open circular, linear and circular dimers and trimers. Examination of DNA from mature phage particles reveals that a circular DNA species is present in at least 0.1 to 1% of the population. The purified complex is extremely efficient in transfection of E. coli spheroplasts. We estimate the molecular weight of the protein in this DNA-protein complex to be approximately 64,000, and suggest that this complex might represent the integrative precursor of infecting Mu DNA.     

Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). I. Undamaged covalent circular lambda DNA molecules in uvrA+ recA+ lysogenic host cells are cut following superinfection with psoralen-damaged lambda phages.

When λ bacteriophages were treated with a photosensitizing agent, psoralen or khellin, and 360 nm light, monoadducts and interstrand crosslinks were produced in the phage DNA. The DNA from the treated phages was injected normally into Escherichia coli uvrA^? (λ) cells and it was converted to the covalent circular form in yields similar to those obtained in experiments with undamaged λ phages. In excision-proficient host cells, however, there was a dose-dependent reduction in the yield of rapidly sedimenting molecules, and a corresponding increase in slow sedimenting material, the extent of this conversion corresponding to about one cut per two crosslinks. Presumably, the damaged λ DNA molecules were cut by the uvrA endonuclease of the host cell, but were not restored to the original covalent circular form.The presence of psoralen damage in λ phage DNA greatly increased the frequency of genetic exchanges in λ phage-prophage crosses in homoimmune lysogens (Lin et al., 1977). As genetic recombination is thought to depend on cutting and joining in DNA molecules, experiments were performed to test whether psoralen-damaged λ DNA would cause other λ DNA in the same cell to be cut. E. coli (λ) host cells were infected with ³²P-labeled λ phages and incubated to permit the labeled DNA to form covalent circles. When these host cells were superinfected with untreated λ phages, there was no effect upon the circular DNA. When superinfected with λ phages that had been treated with psoralen and light, however, many of the covalent circular molecules were cut. The cutting of undamaged molecules in response to the damaged DNA was referred to as “cutting in trans”. It required the uvrA⁺ and recA⁺ host gene functions, but neither recB⁺ nor any phage gene functions. It occurred normally in non-lysogenic hosts treated with chloramphenicol before infection. Cutting in trans may be one of the steps in recA-controlled recombination between psoralen crosslinked phage λ DNA and its homologs.     

Abortive Infection of Bacillus subtilis Bacteriophage PBS1 in the Presence of Actinomycin D

Actinomycin D caused the irreversible loss of PBS1 phage infectious centers and PBS1-mediated transductants. The loss of infectious centers occurred only within the first 4 min after the addition of phage to cells. Actinomycin did not inactivate free phage or inhibit phage adsorption. Electron micrographs indicated that phage adsorbed to cells in the presence of actinomycin ejected their deoxyribonucleic acid (DNA) normally. However, when cells were infected in the presence of actinomycin, 15 to 22% of their (32)P-labeled DNA appeared in the medium, whereas only 1.5 to 7.2% of the (32)P-labeled DNA appeared in the medium during normal infection. Neither 8-azaguanine nor chloramphenicol caused a similar loss of PBS1 infectious centers or transductants. Actinomycin also caused the loss of SP10 infectious centers but it had no effect on SP01 or phi29 infections. We conclude that actinomycin causes abortion of PBS1 infection by inhibiting the uptake or retention of phage DNA into host cells. The immunity of SP01 and phi29 infections to actinomycin probably reflects differences in the penetration mechanisms of these phages.     

Degradation of the viral strand of phiX174 parental replicative-form DNA in a rep- host.

A progressive degradation of the parental viral strand label is observed upon infection of a Rep- mutant of Escherichia coli by 32P-labeled phiX174. Very little parental label remains in the RF (replicative form) by 47 min after infection. Concomitant with this degradation, replicative intermediates are formed which sediment at 21s, the rate of RF I (supercoiled-closed circular DNA), in a neutral sucrose gradient but which denature and sediment in alkaline gradients as single strands of unit size and larger. These denaturable 21s replicative intermediates have been shown previously to be RF molecules containing an elongated viral strand. Addition of chloramphenicol at 7 min after infection at 30 mug/ml, a concentration sufficient to block RF leads to SS (single strand) synthesis but not RF leads to RF synthesis in a wild-type host cell, reduced the amount of viral strand elongation but did not prevent viral strand degradation. The addition of chloramphenicol at 150 mug/ml at 7 min after infection totally prevents both the degradation of the parental label and the formation of the replicative intermediates with elongated tails. We infer that degradation of the viral strand requires the gene A-mediated nicking of the viral strand but not the concomitant elongation of the viral strand.     

Continued Synthesis of Bacterial DNA After Infection by Bacteriophage T4

Early in infection by bacteriophage T4, before replication has commenced, one can detect the presence of newly synthesized DNA which cosediments with parental phage DNA on sucrose gradients. As shown earlier (R. E. Murray and C. K. Mathews, 1969), some of this represents covalent attachment of new material to parental phage DNA molecules. However, as shown herein, most of it is bacterial DNA, which is synthesized after infection and presumably degraded to T4 DNA-sized pieces. The small amount of phage-specific DNA synthesis which occurs is apparently a repair process, for its extent is greatly increased if the phage are irradiated with ultraviolet light prior to infection. Analysis by means of pulse labeling with [(3)H]thymidine and DNA-DNA hybridization shows that host DNA synthesis continues at a significant rate (40 to 80% of the preinfection rate) as late as 10 min after infection at 37 C. Very early in infection this is primarily replicative synthesis, but later a repair process predominates. Presumably this represents attempted repair of damage being inflicted on host DNA by phage-coded nucleases.     

Rapid Method To Characterize Lactococcal Bacteriophage Genomes

We present a rapid method to isolate and analyze bacteriophage DNA. Cells are infected and phage replication is allowed to proceed normally for 30 to 60 min. Prior to DNA packaging and cell bursts, the infected cells (1 ml) are harvested and lysed by using a combination of lysozyme and sodium dodecyl sulfate treatments. The total DNA recovered is enriched for phage genomes, and restriction fragments of the phage DNA can be readily visualized on agarose gels. This method was used to grossly compare the genomes of nine lactococcal phages isolated from different cheese plants at different times. The method was also used to visualize the inhibitory effects of pTR2030-induced abortive infection on the replication of phage nck202.31 in its homologous host, Lactococcus lactis NCK203.     

Gin-mediated site-specific recombination in bacteriophage Mu DNA: overproduction of the protein and inversion in vitro

Inversion of the G segment in bacteriophage Mu DNA occurs by a site-specific recombination event and determines the host specificity of Mu phage particles produced. Inversion is mediated by a Mu function (Gin). The gin gene has been placed under control of the inducible λ pL promoter and a synthetic Shine-Dalgarno linker upstream of the initiation codon. The Gin protein content in induced cells is boosted to ˜10% of total protein. Partially purified extracts from overproducing strains promote efficient inversion of the G DNA segment in vitro which is visualized by agarose gel electrophoresis of the substrate DNA after cutting with appropriate restriction endonucleases. The in vitro reaction requires Mg²⁺, a super-coiled DNA substrate and occurs in the absence of exogenous ATP. Inversion from the G(+) to the G(−) orientation is as efficient as the switch from G(−) to G(+).     

Inhibition of bacterial segregation by early functions of phage Mu and association of replication protein B with the inner cell membrane

Summary Infection of Mu-sensitive bacteria with a recombinant phage that carries the EcoRI·C fragment from the immunity end of wild type Mu DNA causes filamentous growth. Transmission electron microscopy revealed that the cell-division cycle was inhibited at, or prior to, the initiation of septation. The filamentation does not occur after infection of Mu-immune bacteria or after infection with a phage carrying the same EcoRI·C fragment, but with an IS1 insertion in gene B of Mu, showing that either gpB and/or some non-essential functions (e.g. kil) mapping downstream from the insertion are required for the inhibition of cell division. These data and previously published evidence suggest that in the killing of E. coli K12 by early Mu functions expressed from the cloned EcoRI·C fragment, two components have to be distinguished: one, a highly efficient elimination of plasmid DNA carrying the early Mu genes, and second, a series of interactions with host functions conducent to an inhibition of cell division. It is suggested that functions normally involved in the SOS reaction participtate in the inhibition of cell division by early Mu functions. Infected bacteria synthesize the replication protein B (M_R 33000) of Mu, which was found by cell fractionation experiments to be associated with the inner cell membrane. The role of this association for filamentous growth and for the integrative replication of the phage is discussed. The recombinant phage might be useful as a tool for the study of the E. coli cell division cycle.     

Chromatin assembly in isolated mammalian nuclei.

Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions.     

Characterization of Infectious Deoxyribonucleic Acid from Temperate Bacillus subtilis Bacteriophage φ 105

Phenol-extracted, infectious deoxyribonucleic acid (DNA) species from phi105 phage particles, from phi105 lysogenic bacteria, and from induced phi105 lysogenic bacteria were sedimented in sucrose gradients. Infectious DNA from phi105 particles sedimented like the bulk of mature phage DNA in neutral sucrose. Infectivity of prophage DNA was associated with fast-sedimenting material of heterogenous size. Infectious vegetative phage DNA sedimented somewhat faster than mature phage DNA; it was rapidly converted to a poorly infectious form during the infection.     

Intracellular Development of Bacteriophage φR: II. Fractionation of Replicative Form Deoxyribonucleic Acid Associated with Rapidly Sedimenting Host Cell Components

When Escherichia coli is infected with bacteriophage phiR, parental deoxyribonucleic acid (the single- or double-stranded DNA containing the isotopic label of the infecting phage) becomes firmly attached to a cellular structure and can be isolated as a rapidly sedimenting component as described earlier for phiX174. If this component is centrifuged to equilibrium, two peaks of infective DNA are observed at densities of 1.30 and 1.15 g/ml. At low multiplicities of infection, (32)P-labeled parental DNA is found associated with only the cellular components in the dense band; as the multiplicities of infection are increased, the dense band becomes saturated and parental DNA molecules are then found at the light density as well. Actively replicating host DNA is found only in the dense band, whereas progeny DNA, which does not replicate semiconservatively, can become associated with cellular components in the light band. This fractionation of cellular components on the basis of their buoyant density separates primary sites of DNA replication associated with the dense band from nonfunctional binding sites in the light band.     

Initiation of recA+-dependent recombination in Escherichia coli (lambda). II. Specificity in the induction of recombination and strand cutting in undamaged covalent circular bacteriophage 186 and lambda DNA molecules in phage-infected cells.

Covalent circular λ DNA molecules produced in Escherichia coli (λ) host cells by infection with labeled λ bacteriophages are cut following superinfection with λ phages damaged by exposure to psoralen and 360 nm light. This cutting of undamaged covalent circular molecules is referred to as “cutting in trans”, and could be a step in damage-induced recombination (Ross &; Howard-Flanders, 1977). Similar experiments performed with the temperate phage 186, which is not homologous with phage λ, showed cutting in trans and damage-induced recombination to occur in homoimmune crosses with phage 186 also. Double lysogens carrying both λ and 186 prophages were used in a test for specificity in cutting in trans and in damage-induced recombination. The double lysogens were infected with ³H-labeled 186 and ³²P-labeled λ phages. When these doubly infected lysogens containing covalent circular phage DNA molecules of both types were superinfected with psoralen-damaged 186 phages and incubated, the covalent circular 186 DNA was cut, while λ DNA remained intact. Similarly, superinfection with damaged λ phages caused λ, but not 186, DNA to be cut. Evidently, cutting in trans was specific to the covalent circular DNA homologous to the DNA of the damaged phages. Homoimmune phage-prophage genetic crosses were performed in the double lysogenic host infected with genetically marked λ and 186 phages. Damage-induced recombination was observed in this system only between the damaged phage DNA and the homologous prophage, none being detected between other homolog pairs present in the same cell. This result makes it unlikely that the damaged phage DNA induces a general state of enhanced strand cutting and genetic recombination affecting all homolog pairs present in the host cell. The simplest interpretation of the specificity in cutting and in recombination is as follows. When they have been incised, the damaged phage DNA molecules are able to pair directly with their undamaged covalent circular homologs. The latter molecules are cut in a recA ⁺ -dependent reaction by a recombination endonuclease that cuts the intact member of the paired homologs.     

A new procedure for the measurement of the protein-kinase-catalyzed incorporation of the gamma-32P-phosphoryl group of ATP into phospho-proteins and phosphopeptides.

Separate estimations of intact apurinic sites and single-strand breaks in DNA necessitates the use of neutral sucrose gradients for sedimentation analysis after denaturation with formamide or with NaOH followed by reneutralization. The number of breaks per strand in the denatured sample, relative to a control, can be determined with the computer program of Gillespie et al. ⁶; the particular equation for denatured DNA in neutral sucrose gradient that relates the molecular weight and the sedimentation rate is given. The reliability of the whole technique was proven in an experiment with T7 phage [³²P]DNA in which the ³²P transmutations into ³²S were the origin of the strand breaks.     

Structure and Function of Bacteriophage R17 Replicative Intermediate Ribonucleic Acid II. Properties of the Parental Labeled Molecule

Replicative intermediate ribonucleic acid (RNA), designated RI, which contained parental RNA labeled with (32)P was separated by filtration through agarose from the nucleic acids prepared from (32)P-labeled RNA phage-infected Escherichia coli. A larger amount of ribonuclease-sensitive parental label was found in the rapidly sedimenting forms of RI than in the slower sedimenting forms, indicating that parental RNA is displaced to form a single-stranded tail. This result indicates that some phage RNA is generated by asymmetric semiconservative replication of RI, but it does not mean that a portion of the RI duplexes cannot be conserved during generation of phage RNA. Parental RNA was also found in double-stranded RNA with no apparent tails which sedimented with an S value of 13. This RNA was soluble in 2 m NaCl, and its sedimentation rate was unaffected by ribonuclease; nevertheless, single-strand scissions were produced by ribonuclease and were detected after the duplex was converted to its component single strands.     

Intermediates in genetic recombination of bacteriophage T7 DNA. Biological activity and the roles of gene 3 and gene 5

When Escherichia coli cells were infected with ³²P- and 5-bromodeoxyuridine-labeled T7 bacteriophage defective in genes 1.3, 2.3, 4 and 5, doubly branched T7 DNA molecules with “H” or “X”-like configurations were found in the half-heavy density fractions. Physical study showed that they are dimeric molecules composed of two parental DNA molecules (Tsujimoto & Ogawa, 1977a). The transfection assay of these molecules revealed that they were infective. Genetic analysis of progeny in infective centers obtained by transfection of dimeric molecules formed by infection of genetically marked T7 phage showed that these dimeric molecules were genetically biparental.To elucidate the roles of the products of gene 3 (endonuclease I) and gene 5 (DNA polymerase) of phage T7 in the recombination process, the ³²P/BrdUrd hybrid DNA molecules which were formed in the infected cells in the presence of these gene products were isolated, and their structures were analyzed. The presence of T7 DNA polymerase seems to stimulate and/or stabilize the interaction of parental DNAs. At an early stage of infection few dimeric molecules were formed in the absence of T7 DNA polymerase, whereas a significant number of doubly branched molecules were formed in its presence. With increasing incubation time, the multiply branched DNA molecules with a high sedimentation velocity accumulated.In contrast to the accumulation of multiply branched molecules in phage with mutations in genes 2, 3 and 4, almost all of the ³²P/BrdUrd hybrid DNA formed in phage with mutations in genes 2 and 4 were monomeric linear molecules. Shear fragmentation of monomeric linear ³²P/BrdUrd-labeled DNA shifted the density of [³²P]DNA to almost fully light density. It was also found that approximately 50% of [³²P]DNA was linked covalently to BrdUrd-labeled DNA. These linear monomer DNA molecules had infectivity and some of those formed by infection of genetically marked parents yielded recombinant phages. Therefore the gene 3 product seems to process the branched intermediates to linear recombinant molecules by trimming the branches.     

Growth of bacteriophage Mu in Escherichia coli dnaA mutants.

In one-step growth experiments we found that bacteriophage Mu grew less efficiently in nonreplicating dnaA mutants than in dnaA+ strains of Escherichia coli. Phage development in dnaA hosts was characterized by latent periods that were 15 to 30 min longer and an average burst size that was reduced by 1.5- to 4-fold. The differences in phage Mu development in dnaA and dnaA+ strains were most pronounced in cells infected at a low multiplicity and became less pronounced in cells infected at a high multiplicity. Many of these differences could be eliminated by allowing the arrested dnaA cells to restart chromosome replication just before infection. In continuous labeling experiments we found that infected dnaA strains incorporated 5 to 40 times more [methyl-3H]thymidine than did uninfected cells, depending on the multiplicity of infection. DNA-DNA hybridization assays showed that greater than 90% of this label was contained in phage Mu DNA sequences and that only small amounts of the label appeared in E. coli sequences. In contrast, substantial amounts of label were incorporated into both host and viral DNA sequences in infected dnaA+ cells. Although our results indicated that phage Mu development is not absolutely dependent on concurrent host chromosomal DNA replication, they did strongly suggest that host replication is necessary for optimal growth of this phage.