Formation of the parental replicative form DNA of bacteriophage phi-X174 and initial events in its replication

Intracellular φX174 DNA was studied under a variety of conditions that prevent the replication of the parental replicative form DNA. These conditions included treatment with 150 μg of chloramphenicol per ml., the use of the rep₃⁻ mutation of the host cell, amber mutation (am 8) in the viral gene responsible for RF replication (gene A) ^† and combinations thereof. In all cases the majority of the parental RF was in the covalently closed form (RFI). The relative amount of RF with a discontinuity in one strand (RFII) in these cases was between 2 and 10% of the total RF and independent of the multiplicity of infection. The only exception was seen in infections of rep₃ cells with φX am 3 (a mutant in the lysis gene, gene E, used as a wild-type representative). In this case a fairly constant absolute amount of RFII (1 to 4 per cell), independent of the multiplicity of infection, was formed, consisting almost exclusively of a closed complementary and an open parental viral strand. Since the formation of this type of RFII was dependent on protein synthesis and the presence of the product of φX gene A, it is concluded that the discontinuity in the parental viral strand represents the result of the action of the gene A product on the DNA. Possible mechanisms for the mode of action of the gene A product are discussed.     

On the molecular length of the replicative form DNA of bacteriophage phiX174

This paper describes an electron microscopic study of the circular replicative form DNA of bacteriophage φX174. The study has been carried out using a preparative technique in which the DNA molecules are adsorbed from solution on to the cleavage surface of mica and visualized in the electron microscope as a metal-shadowed replica (Gordon &; Kleinschmidt, 1969,1970). Contour lengths of open circular molecules were measured in samples obtained from preparations in which the following experimental parameters were varied: the ionic strength of the solution from which the DNA was adsorbed on the mica and the way in which the molecules were dried before shadowing. At the 0.05 significance level, varying these parameters had no effect on the mean length and variances of samples of molecules obtained from five experiments; the samples were therefore regarded as being drawn from the same molecular population with a mean length and variance of, respectively, 1.83 μm and 0.0117 μm².It was argued that the DNA molecules adsorbed on the mica are “frozen” into the molecular conformation present in solution at the time of adsorption and that, therefore, the experimentally determined contour lengths represent authentic molecular lengths in solution. Based on current estimates of the replicative form DNA molecular weight, the mean contour length obtained was slightly but significantly larger than the length predicted for molecules in an exact B configuration. The variance was larger than could be attributed solely to experimental error, indicating that the molecular population in aqueous solution is heterogeneous in contour length. These experimental results were shown to be consistent with a model for DNA structure in aqueous solution in which individual molecules are dynamic variants of a perturbed B form structure (von Hippel &; Wong, 1971).     

A rolling circle model of the in vivo replication of bacteriophage phiX174 replicative form DNA: different fate of two types of progeny replicative form

In a preceding paper (Schröder and Kaerner, 1972) a rolling circle mechanism has been described for the replication of bacteriophage φX174 replicative form. Replication involved nicking and elongation of the viral (positive) strand component of the RF molecule resulting in the displacement of a single-strand tail of increasing length. The synthesis of the new complementary (negative) strand on the single-strand tails appears to be initiated with considerable delay and converts the tail into double-stranded DNA. Before the new negative strand is completed the replicative intermediates split into (I) a complete RF molecule containing the “old” negative and the new positive strand, and (II) a linear, partially double-stranded “tail” consisting of the complete old positive strand and a fragment of the new negative strand.The present study is concerned with the fate during RF replication of these fragments of the rolling circles. Those RFII molecules containing the old negative strands appear to go into further replication rounds repeatedly. Some of the tails were found in the infected cells in their original linear form. “Gapped” RFII molecules, which have been described earlier by Schekman and co-workers (Schekman &; Ray, 1971; Schekman et al., 1971), are supposed to originate from the tails of rolling circle intermediates by circularization of their positive strand components. Evidence is provided by our experiments that even late during RF replication these gaps are present only in the negative strands of RFII. Appropriate chase experiments indicated that the tails finally are converted to RFI molecules. Progeny RFI molecules could not be observed to start new replication rounds under our conditions although we cannot exclude that this might happen to some minor extent.The results presented suggest that the master templates for RF replication are the first negative strands to be formed, rather than the parental positive strands.