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. 1981 Mar;78(3):1476–1480. doi: 10.1073/pnas.78.3.1476

Multiple rounds of adenovirus DNA synthesis in vitro

Marshall S Horwitz 1,2,3, Hiroyoshi Ariga 1,2,3
PMCID: PMC319153  PMID: 6262804

Abstract

Adenovirus (Ad) type 2 DNA synthesis can be initiated in the presence of a soluble extract of uninfected HeLa cell nuclei, a 25-60% saturated ammonium sulfate fraction of infected cytoplasm and viral DNA covalently linked to a 5′-terminal protein (Ad DNA-prot). As the purification, from either the nuclei or cytoplasm, of factors active in DNA replication proceeded, various nonreplicative reactions which incorporate labeled deoxynucleotides were uncovered. In order to distinguish replicative from repair reactions, an assay was developed in which the Ad DNA-prot was digested with Xba I, all of the fragments so produced were used (without separation) in a replication reaction, and the products were assayed by electrophoresis on neutral agarose gels. In replicative reactions, most of the radioactivity was incorporated into the terminal fragments, with the internal fragments remaining unlabeled. Infected cytoplasm contains a “discrimination” function in addition to specific factors for Ad DNA replication. The discrimination factors inhibit the nonspecific nucleotide incorporation by uninfected HeLa nuclear extracts on Ad DNA-prot. The specific replicative incorporation into the terminal Ad DNA-prot fragments has also allowed an independent assay for reinitiation of progeny molecules synthesized in vitro. After the first round of replication, the 5′ strand of the progeny duplex from each end is labeled. These same labeled strands will be displaced during the second round of replication and appear in new bands which have been shown to be the single-strand equivalents of the terminal fragments. Thus, at least two rounds of Ad DNA synthesis can initiate at each terminus in vitro. The appearance of displaced single strands requires DNA replication because the addition of dideoxycytidine triphosphate after the first round of synthesis prevents the displacement reaction. Both the progeny single- and double-stranded DNA appear to be linked to protein.

Keywords: eukaryotic DNA replication, soluble nuclear extracts, soluble cytoplasmic extracts, DNA-protein interaction

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