The mechanism of replication of φX174 DNA: XVI. Evidence that the φX174 viral strand is synthesized discontinuously

Abstract

Using a new method of stopping DNA synthesis, we have studied the nascent intermediates present during the final stage of φX174 DNA replication when progeny single-stranded circular DNA molecules are synthesized. We find that 40 to 50% of the [ 3H]thymidine incorporated in a brief pulse, stopped by bringing the infected culture rapidly to 100 °C, is present in DNA molecules shorter than unit length. The molecules, which range from very short to unit length, are not generated by the stopping and isolating procedure, since 32P-labeled infecting parental viral strands remain relatively intact. The proportion of pulse label found in short intermediates varies with pulse length, stopping procedure, aeration level of the infected culture, and host strain. There is no significant difference in the abundance of short nascent intermediates in ung and ung + strains, suggesting that the short molecules are not the result of uracil excision by uracil-DNA glycosylase. The 3H-labeled short molecules hybridize to all regions of the φX genome, but preferentially to the region around the origin/terminus of replication. The relative amount of 3H in the unit length molecules that hybridizes to restriction enzyme fragments increases from the origin to the terminus, indicating that these molecules were completed during the pulse interval. Sensitivity to spleen exonuclease after treatment with alkali or RNase suggests that some of the short molecules isolated both during viral strand synthesis and during replicative form DNA replication have at least one ribonucleotide at the 5′ end. We conclude that the major mode of φX viral strand DNA replication is a discontinuous process, not continuous as proposed by the rolling circle model.