Studies on the mechanism of replication of adenovirus DNA. III. Electron microscopy of replicating DNA

Replicating Ad5 DNA was isolated from nuclei of infected KB cells and studied by electron microscopy. Branched as well as unbranched linear intermediates were observed containing extended regions of single-stranded DNA. The relationship between the branched and unbranched structures was studied during synchronized synthesis in the first round of replication after release of hydroxyurea inhibition. The branched intermediates represented early and the unbranched intermediates late stages in the replication cycle. Digestion of the branched intermediates with Eco RI endonuclease revealed that replication had started at the molecular right end (the A-T-rich end). This was confirmed by partial denaturation mapping of branched intermediates. These results confirm and extend a tentative model on the mechanism of Ad5 DNA replication