The Process of Infection with Bacteriophage φX174: XVI. Synthesis of the Replicative Form and its Relationship to Viral Single-Stranded DNA Synthesis

The incorporation of ^3H-labeled thymine or thymidine into the DNA of mitomycin C-pretreated bacteria was studied at various times during their infection with bacteriophage φX174am3 (a lysis-defective mutant). Experiments in which the labeled DNA precursor was present from the onset of infection showed that net synthesis of the replicative form of φX DNA ceases between 15 and 20 minutes after infection. However, administration of the labeled DNA precursor in short pulses at times later than 20 minutes of infection (30ºC), demonstrated incorporation of label into RF-DNA§ (RFI and RFII), as well as into SS-DNA. Upon use of even shorter pulses (30 to 60 seconds), the label was incorporated almost exclusively into RFII. Since these pulses were administered at times of maximum SS-DNA synthesis, it would follow from these and other results that the synthesis of RF-DNA, in particular RFII, is an intermediate step in the synthesis of SS-DNA. Since there appears to be no net synthesis of RF after 15 to 20 minutes of infection, there must be a turnover of RF-DNA accompanying SS-DNA synthesis. To test directly whether RFII serves as a precursor for SS-DNA, a pulse and chase experiment was performed. The result showed that label present in RFII before the chase appeared as SS-DNA after the chase. A similar experiment showed that early RF-DNA (mostly RFI) labeled between zero and seven minutes of infection also serves as precursor for SS-DNA. However, the rate of conversion of early RF-DNA appears to be lower than the rate of conversion of late RFII. These results suggest that RFII is the direct precursor of viral SS-DNA