Vegetative bacteriophage λ DNA II. Physical Characterization and Replication

Abstract

The structure and replication of the fast-sedimenting form of λ DNA found after infection have been examined. The sedimentation rate at neutral and alkaline (strand-separating) pH, shear resistance, and buoyant density in alkaline CsCl are consistent with the interpretation that the rapidly sedimenting DNA is a covalently-closed, circular duplex wound in a superhelix. Mitomycin C has been used to inhibit bacterial DNA synthesis in Escherichia coli hcr− cells infected with λ. An analysis of the λ DNA synthesized under these conditions reveals that before intracellular phage are produced, synthesis of DNA with the sedimentation properties of the closed circular form predominates. At the end of the phage eclipse period, net synthesis of circular DNA stops and synthesis of DNA with the sedimentation properties of mature viral DNA begins. If bacteria are infected with λ in the presence of 100 μg of chloramphenicol/ml. the conversion of the injected phage DNA to the fast-sedimenting form occurs, but no replication ensues. In the presence of 30 μg of chloramphenicol/ml. (added 15 minutes before infection), the circular DNA is able to replicate, albeit more slowly than in the absence of chloramphenicol. The synthesis of DNA with the sedimentation properties of mature viral λ DNA is completely inhibited in the presence of 30 μg of chloramphenicol/ml. The replication of the circular DNA has been studied using density and radioactivily labeled phage particles to initiate the infection. The circular DNA of parental origin consists of both unreplicated and semi-conservatively replicated molecules. If the infection is performed in medium containing [3H]thymine, the progeny circular DNA has either a hybrid density or a fully light density. The ratio of progeny to parental DNA rises to at least 20 by the end of the phage eclipse period. Most closed-circular DNA molecules containing parental label replicate more than once semi-conservatively. The DNA of hybrid density derived from the fast-sedimenting component on a sucrose gradient is resistant to strand separation in alkali. The formation of parental and progeny circular DNA explains the eclipse of infectivity measured in the helper assay (Dove & Weigle, 1965).

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