Adenovirus DNA replication in vivo: properties of short DNA molecules extracted from infected cells

Adenovirus type 5 (Ad5) DNA replicating in intact HeLa cells was pulse-labeled with [3H]thymidine extracted by the procedure of Hirt and analyzed on neutral sucrose gradients. In addition to viral replicative forms (over 33 kb), slowly sedimenting species of DNA (0.05-3 kb) was observed. Hybridization analysis showed that this DNA contained about 30% Ad5 DNA sequences. Analysis of the sensitivity of this DNA to the 5' OH-specific spleen exonuclease after alkali or RNAase treatment revealed that about 80% of these molecules contained ribonucleotides. DNA in this fraction was labeled at the 5' end with 32P and hybridized together with control 3H-labeled Ad5 DNA to Hpa I restriction fragments immobilized on nitrocellulose paper. Many of these small molecules were located near the termini. Alkali treatment prior to hybridization decreased the 32P/3H ratio throughout the genome. This suggests that some of these Ad5 molecules possess ribonucleotides and therefore may be intermediates in discontinuous DNA replication. Longer molecules (over 0.5 kb) were found sedimenting with viral replicative forms and mature DNA. The Ad5 molecules in this fraction showed no evidence of alkali-labile termini.