Deletion Mutants of Bacteriophage ϕX174

Mutants of bacteriophage ϕX174 have been isolated that are less dense than wild-type ϕX particles in CsCl. When mutant viral (+) strand DNA and wild-type complementary (-) strand DNA are hybridized, the resulting duplex molecules have single-stranded loops characteristic of wild-type-deletion heteroduplexes. The mutant bacteriophages fail to complement ϕX amber mutants in cistron E. We conclude that the mutant viruses have deleted approximately 7% of the ϕX genome in the region of cistron E.     

Replication of ϕX174 DNA by Escherichia coli polA-In Vitro

Lysates of an Escherichia coli polA^- strain convert single-stranded DNA from ϕX174 virus to the double-stranded replicative form with high efficiency on cellophane discs. The initiation of synthesis of the complementary strand appears to be rate limiting; once initiation occurs, the chain is propagated rapidly. Under these conditions, the unsealed replicative form accumulates and is slowly converted to the sealed form; this conversion requires the activity of the DPN-dependent DNA ligase.     

Pyrimidine Sequences from the DNA of Bacteriophages fd, fl, and ϕX174

By the use of techniques previously developed to determine the sequence of large, unique pyrimidine oligonucleotides in bacteriophage fd DNA, pyrimidine sequences in the related phage fl and the unrelated phage varphiX174 amber 3 were determined. The DNA of fl contains many pyrimidine sequences completely identical to those in fd, including one sequence of 20 base residues. The large pyrimidine oligonucleotides in varphiX174 DNA are quite different from those in fd, although some homology is observed. There is present in each of the phage DNAs a pyrimidine oligonucleotide rich in thymidine that contains a common sequence of C_T_T_T_T_T_T_T.     

STUDIES ON THE PROTEINS OF ΦX174, II. THE PROTEIN COMPOSITION OF THE ΦX COAT

In order to determine the composition of the PhiX174 coat, PhiX particles labeled in most of the amino acids have been dissociated, and the proteins separated by electrophoresis on polyacrylamide gels. The molar ratios of the four proteins calculated from the fraction of radioactivity in each peak and the molecular weights of the individual proteins suggest that the capsid is composed of 60 molecules of the 48,000 daltons protein and that each of the 12 spikes is composed of one molecule of the 36,000 daltons protein and five each of the 19,000 and 5,000 daltons proteins.     

ϕX174 cistron A protein is a multifunctional enzyme in DNA replication

The cistron A protein induced by phage varphiX174 nicks (produces a single-strand break in) the viral strand of the superhelical varphiX duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA in the electron microscope, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the varphiX map [Sanger, F., Air, G. M., Barrel, B. G., Brown, N. L., Coulson, A. R., Fiddes, J. C., Hutchison, C. A., III, Slocomb, P. M. Y. & Smith, M. (1977) Nature 265, 687-695]. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication.The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP, and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles per cisA-DNA complex.The replicating intermediates seen in the electron microscope are a novel form of "rolling circle" [Gilbert, W. & Dressler, D. H. (1969) Cold Spring Harbor Symp. Quant. Biol. 33, 473-485]. The 5' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: (i) nicking the viral strand at the origin of replication to initiate a round of replication, (ii) participating in a complex which supports fork movement in strand separation and replication, (iii) nicking again at the regenerated origin to produce a unit-length DNA, and (iv) ligating the newly generated 3'-OH end to the 5'-phosphate-complexed end to form a circular viral molecule.     

Replicative Form II DNA of ϕX174; Resistance to Exonucleolytic Cleavage by E. coli DNA Polymerase

The progeny replicative form II DNA isolated from E. coli cells infected with ϕX174 is resistant to the exonucleolytic activity associated with E. coli DNA polymerase. A limited endonucleolytic cleavage with micrococcal endonuclease renders the replicative form II molecule susceptible to the exonucleolytic activity associated with the E. coli DNA polymerase.     

Conversion of ϕX174 Viral DNA to Double-Stranded Form by Purified Escherichia coli Proteins

The E. coli proteins that catalyze the conversion of varphiX174 single-stranded DNA to duplex DNA have now been purified extensively. The reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, DNA elongation factors I and II, E. coli DNA binding protein, and two additional E. coli proteins, replication factors X and Y. DNA synthesis by these proteins requires varphiX174 viral DNA, dNTPs, Mg(+2), and ATP. The product synthesized is full-length linear varphiX174 DNA. The reaction has been resolved into two steps. The first step involves the interaction of ATP and varphiX174 DNA with dnaB and dnaC(D) gene products, E. coli DNA binding protein, and replication factors X and Y in the absence of dNTPs. Subsequent dNMP incorporation requires the addition of DNA polymerase III, DNA elongation factors I and II, dnaG gene product, and dNTPs.     

EVENTS OCCURRING DURING THE REPLICATION OF BACTERIOPHAGE T4 DNA

Ten temperature-sensitive mutants of T4D have been examined to find the times in the lytic cycle at which a shift to a restrictive temperature no longer exerts a lethal effect. Nine of these mutants play a role in phage DNA synthesis. The results of these experiments suggest that the products of three of the genes tested—genes 42, 44, and 45—have control or complexing functions in addition to any enzymatic ones. The data also indicate that the products of genes 30 and 41 may act cooperatively. The properties of mutants of gene 43 (DNA polymerase) and gene 56 (dCTP-dUTPase) suggest that the production of a certain length of concatameric DNA is necessary before viable phage can be produced.     

Initiation of DNA Synthesis: Synthesis of ϕX174 Replicative Form Requires RNA Synthesis Resistant to Rifampicin

Conversion of single-stranded DNA of phage ϕX174 to the double-stranded replicative form in Escherichia coli uses enzymes essential for initiation and replication of the host chromosome. These enzymes can now be purified by the assay that this phage system provides. The ϕX174 conversion is distinct from that of M13. The reaction requires different host enzymes and is resistant to rifampicin and streptolydigin, inhibitors of RNA polymerase. However, RNA synthesis is essential for ϕX174 DNA synthesis: the reaction is inhibited by low concentrations of actinomycin D, all four ribonucleoside triphosphates are required, and an average of one phosphodiester bond links DNA to RNA in the isolated double-stranded circles. Thus, we presume that, as in the case of M13, synthesis of a short RNA chain primes the synthesis of a replicative form by DNA polymerase. Initiation of DNA synthesis by RNA priming is a mechanism of wide significance.     

The Rolling Circle for ϕX DNA Replication, II. Synthesis of Single-Stranded Circles

ϕX-infected cells have been allowed to incorporate tritiated thymidine late in the phage life cycle when single-stranded circles are the product of DNA synthesis. Virtually all of the radioactivity is recovered in a continuum of actively replicating viral DNA molecules. These molecules are termed rolling circle intermediates because they are characterized by three structural properties. They possess positive strands that are longer than the length of a mature viral genome, and negative strands that are covalently closed single-stranded circles. The 3′ termini of the long positive strands lie upon the template rings, while the 5′ ends are free in solution.     

The Rolling Circle for ϕX DNA Replication, III. Synthesis of Supercoiled Duplex Rings

During varphiX duplex ring synthesis, the first supercoils to acquire radioactivity after the addition of tritiated thymidine are labeled only in their negative strands. In longer pulses, this asymmetry of labeling progressively disappears. This finding supports the rolling circle model for DNA replication due to the structural asymmetry of its replicating intermediate, but is not predicted by the Cairns or Yoshikawa models.     

Cis-Limited Action of the Gene-A Product of Bacteriophage ϕX174 and the Essential Bacterial Site

Parental replicative-form (RF(*)) DNA of bacteriophage varphiX174 in a replication-deficient host cell (rep(3) (-)) exhibits two characteristic features that correlate the function of viral gene A with the initiation of viral DNA replication: a specific discontinuity in the viral strand of a constant number of RF molecules and elongation of the viral strand to yield replicative-intermediate DNA forms with single-stranded tails. At high multiplicities of infection, these initiation events are limited to an average of four specifically nicked RFII molecules per cell. The limiting factor from the host cell may be related (or identical) to the essential bacterial sites known to limit the participation of parental genomes in RF replication. Double-infection experiments with wild-type phage and phage carrying an amber mutation in gene A show that the formation of gene A-specific RFII and RI is cis-limited to only the wild-type DNA. These results provide a basis at the DNA level for the known asymmetric complementation of gene A.     

DNA-DEPENDENT RNA-DIRECTED PROTEIN SYNTHESIS in vitro, II. SYNTHESIS OF A ϕX-174 COAT PROTEIN COMPONENT

We present evidence to show that the primary protein product of an in vitro DNA-dependent RNA-directed protein-synthesizing system primed by varphiX-174 replicative form DNA comigrates in a gel electrophoresis system with a phage structural component. This protein component is precipitable by antiserum to purified phage.     

Electron Microscope Studies of Heteroduplex DNA from a Deletion Mutant of Bacteriophage ϕX-174

A population of double-stranded replicative form of DNA molecules from bacteriophage varphiX-174 carrying a deletion of about 9% of the wild-type DNA has been discovered in a sample cultivated under conditions where the phage lysozyme gene is nonessential. The structures of deleted monomers, dimers, and trimers were studied by the electron microscope heteroduplex method. The dimers and trimers are head-to-tail repeats of the deleted monomers. Some interesting examples of the dynamical phenomenon of branch migration in vitro have been observed in heteroduplexes of deleted dimer and trimer strands with undeleted monomer viral strands from the wild-type phage.