DNA synthesis in nucleotide-permeable Escherichia coli cells : II. Synthesis of replicative form DNA of phage φX174

Abstract

Escherichia coli cells were treated with mitomycin C to suppress host DNA synthesis, infected for five minutes with bacteriophage φX174, made permeable to nucleotides by treatment with ether and allowed to synthesize DNA from exogenous deoxynucleoside triphosphates. The product was 70 to 80% replicative form (RF) DNA of the phage according to sedimentation and annealing properties. RF synthesis occurred at the normal level in DNA polymerase-deficient cells and did not occur in these cells when they were infected with a gene VI amber mutant of the virus, which in the living cells does not replicate beyond the first RF. The structure of intermediates of RF synthesis in ether-treated cells is thought to be basically consistent with the rolling circle model of RF replication proposed by Knippers, Whalley & Sinsheimer (1969). In addition, the results suggest that a temporary interruption exists in the complementary strand just ahead of the growth point. Complementary strand material, and possibly also viral strand material, exists early after synthesis as short pieces having only a small fraction of the viral length. These pieces are subsequently joined together but can be induced to accumulate. This accumulation is suppressed by 0.2 m m-dATP but not detectably by any of the other deoxynucleoside triphosphates in high concentration, or by ATP or DNA ligase cofactor. Label in RF can be chased up to 80% into supertwisted molecules.