DNA synthesis in nucleotide-permeable Escherichia coli cells : IV. Mode of φX174 replicative form DNA synthesis and the template involved

Abstract

The synthesis of bacteriophage φX174 double-stranded replicative form DNA was studied in nucleotide-permeable Escherichia coli cells prepared from cultures in the RF ¶ ¶ Abbreviations used: RF, double-stranded replicative form DNA; RFI, the supertwisted DNA duplex; RFII, the open-stranded DNA duplex; SS, the single-stranded viral DNA; TT-RF and BB-RF, RF containing thymine or 5-bromouracil, respectively, in both strands; BT-RF, RF containing 5-bromouracil in the viral strand and thymine in the complementary strand; TB-RF, RF containing thymine in the viral and 6-bromouracil in the complementary strand. multiplication stage of the viral infection. Newly synthesized DNA was density-labelled with dBrUTP, or in 13C 15N-substituted cells, with light atoms and studied after the synthesis had levelled off. Some 70% of the dBrUTP-labelled RF DNA was found about equally distributed between the two classes of hybrid RF in which either the viral or the complementary strand is labelled. In the remainder of the labelled RF molecules, the fraction of newly synthesized DNA was lower than 50% according to buoyant density and no significant amounts of fully synthetic RF were found, either in the experiments with dBrUTP or those with 13C 15N-substituted cells. On the basis of various considerations, it is argued that hybrid RF results from semi-conservative replication of both strands in the template duplexes and that asymmetrical synthesis (displacement or repair) is less likely. The number of hybrid RF is estimated to be about 6 per cell under favourable conditions. Both progeny RF and parental RF appear to participate in this synthesis.