Formation of the parental replicative form DNA of bacteriophage φX174 and initial events in its replication

Abstract

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 rep 3 − mutation of the host cell, amber mutation ( am 8) in the viral gene responsible for RF replication (gene A) † † Abbreviations used: Cam, chloramphenicol; [ 3H]dThd, tritiated thymidine; RFI, replioative form DNA with both strands closed; RFII, replicative form DNA with one or more discontinuities in either strand; RI, replicative intermediate DNA; SDS, sodium dodecyl sulfate. 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 rep 3 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. Intermediates during the synthesis of the first complementary strand were isolated from cells infected with ultraviolet light-irradiated phages. Such intermediates contained incomplete linear complementary strands and circular parental viral strands. It is therefore concluded that the virus-specified discontinuity in RFII is introduced after the first complementary strand is completed and closed. Some residual virus-specific DNA synthesis beyond the formation of the parental RF was seen in rep 3 − cells in infections where the viral gene for RF replication was functioning. This residual synthesis resulted in replicative intermediate structures containing circular complementary strands and elongated linear viral strands. The significance of this type of RI for the existing models of φX174 RF replication is discussed.