The rolling circle for phiX DNA replication. II. Synthesis of single-stranded circles.

varphiX-infected cells have been allowed to incorporate tritiated thymidine late in the phage life cycle when single-stranded circles are the product of DNA synthesis. Virtually all of the radioactivity is recovered in a continuum of actively replicating viral DNA molecules. These molecules are termed rolling circle intermediates because they are characterized by three structural properties. They possess positive strands that are longer than the length of a mature viral genome, and negative strands that are covalently closed single-stranded circles. The 3' termini of the long positive strands lie upon the template rings, while the 5' ends are free in solution. From these experimental data, the basic mode of synthesis is deduced to involve the continuous elongation of the open positive strand by endless copying around the circular negative strand template. As new bases are added to the template-bound (3') end of the positive strand, the distal (5') end is displaced from the template ring as a single-stranded tail of increasing length. It is the tail which serves as the source of material for progeny chromosomes. These data confirm our characterization of this varphiX intermediate, which initially was based only on the possession of long positive strands, and extend this characterization to include experimental statements about the circular nature of the template DNA strand, and the 5' to 3' direction of polynucleotide chain growth within the intermediate. Moreover, the description can now be applied to all of the molecules which acquire label during a pulse.