ADENOVIRUS DNA-REPLICATION INVITRO

A soluble extract from the nuclei of HeLa cells infected with adenovirus 5 (Ad5) carries out the semiconservative replication of exogenously added Ad5DNA in vitro. Maximal DNA synthesis is observed when DNA-protein complex, isolated from Ad virions, is added as template. DNA-protein complex form virions of the closely related virus, adenovirus 2, is also active. In contrast, very little in vitro DNA synthesis is observed when deproteinized Ad5 DNA or DNA from a heterologous source (bacteriophage T7) is added as template. The product of the in vitro reaction consists of long Ad5 DNA strands that are hydrogen-bonded, but not covalently linked, to the input DNA template. During the course of the in vitro reaction, branched molecules with structural features identical to in vivo replication intermediates are formed. These findings support the conclusion that replication in the in vitro system closely resembles adenovirus DNA replication in vivo. The system provides as assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.