Replication of vaccinia DNA in mouse L cells III. Intracellular forms of viral DNA

Parental and replicating vaccinia DNA molecules, labeled with [³H]thymidine or [¹⁴C]thymidine, present in infected L cells were analyzed by sedimentation in neutral and alkaline sucrose gradients. Alkaline sucrose gradient sedimentation analysis of labeled parental genomes present in infected cells showed that: (a) Such genomes were not degraded to acid-soluble products during the infection cycle; (b) cell-associated, cross-linked parental molecules (102 and 90–92 S) were “nicked”; parental DNA molecules sedimenting at 70–72 S were detected, but further nicking or degradation did not occur; and (c) molecules sedimenting at 90–92 S appeared to accumulate in the cytoplasm of infected cells and could serve as the templates for semiconservative DNA replication. When analyzed in neutral sucrose gradients, about 90% of viral DNA molecules labeled from 1 to 2 hr postinfection were associated with large aggregates or complexes which pelleted under the conditions of analysis used in these studies. With time (2–3 hr) after infection, viral DNA molecules could be dissociated from such aggregates and resolved by sedimentation in neutral sucrose gradients. The dissociation of labeled viral DNA molecules from complexes required continuous protein synthesis. Viral DNA could be released from complexes by treatment with alkali or digestion with S-1 nuclease, but not with RNase or Pronase. The results suggest that single-stranded (ss) DNA regions per se or proteins having affinity for ssDNA may bind the replicating vaccinia DNA molecules together in complexes.