The structure of replicating adenovirus 2 DNA molecules

Adenovirus 2 (Ad2)-infected KB cells were exposed to a 2.5 min pulse of ³H-thymidine at 19 hr after infection. The labeled DNA molecules were separated from cell DNA and mature Ad2 DNA by sucrose gradient sedimentation and CsCI equilibrium centrifugation under conditions designed to minimize branch migration and hybridization of single strands. Electron microscopy-of fractions containing radioactivity revealed two basic types of putative replicating molecules: Ad2 length duplex DNA molecules with one or more single-stranded branches (type I) and Ad2 length linear DNA molecules with a single-stranded region extending a variable distance from one end (type II). Length measurements, partial denaturation studies and 3′ terminal labeling experiments were consistent with the following model for Ad2 DNA replication. Initiation of DNA synthesis occurs at or near an end of the Ad2 duplex. Following initiation, a daughter strand is synthesized in the 5′ to 3′ direction, displacing the parental strand with the same polarity. This results in the formation of a branched replicating molecule (type I). Initiations at the right and left molecular ends are approximately equal in frequency, and multiple initiations on the same replicating molecule are common. At any given displacement fork in a type I molecule, only one of the two parental strands is replicated. Two nonexclusive mechanisms are proposed to account for the replication of the other parental strand. In some cases, before completion of a round of displacement synthesis initiated at one end of the Ad2 duplex, a second initiation will occur at the opposite end. In these doubly initiated molecules, both parental strands serve as templates for displacement synthesis. Two type II molecules are generated when the oppositely moving displacement forks meet. Alternatively, displacement synthesis may proceed to the end of the Ad2 duplex, resulting in the formation of a daughter duplex and a parental single strand. Replication of the displaced parental strand is then initiated at or near its 3′ terminus, producing a type II molecule. Daughter strand synthesis proceeds in the 5′ to 3′ direction in type II molecules generated by either mechanism, and completion of synthesis results in the formation of a daughter duplex.