REPLICATION OF ADENOVIRUS DNA IN VITRO

Abstract

This thesis describes the development and characterization of two systems for the replication of adenovirus DNA in vitro. Ammonium sulfate (AS) extraction of nuclei from adenovirus-infected cells solubilizes approximately 25% of the viral replicating DNA, leaving the HeLa chromatin behind. AS extracts are capable of synthesizing full-sized adenovirus DNA by elongation of previously-initiated molecules. The direction and termini of replication of progeny DNA strands in vitro are the same as in vivo. The replication activity is associated with large complexes which can be purified away from the bulk of DNA polymerase present in the extracts.;Extracts prepared from cells infected with the temperature-sensitive mutant H5ts107 exhibited temperature-sensitive replication in vitro. Extracts are five times as active in reactions at 30(DEGREES) as they are at 38(DEGREES). Wild type extracts are equally active at the two temperatures. H5ts107 DNA synthesis can be restored to 75% of the 30(DEGREES) level by adding purified adenovirus-coded DNA binding protein to yield full-sized products. DNA binding protein from H5ts107-infected cells failed to restore activity in the mutant extracts at 38(DEGREES).;Replication of exogenously added adenovirus DNA molecules has been studied using virus-infected nuclear extracts described by Challberg and Kelly. Hydroxyurea (HU) extracts catalyze the synthesis of full-sized DNA using template adenovirus DNA-protein complex purified from virions. The origins and direction of chain elongation in this system are identical to those observed in vivo. Replication requires a DNA template bound to the 5' terminal protein and is inhibited by denaturation of this protein. It is inhibited by aphidicolin, an inhibitor of DNA polymerase (alpha), and 2',3'-dideoxythymidine triphosphate, an inhibitor of polymerases (beta) and (gamma).;A sensitive assay for the presence of the 5' terminal protein on adenovirus DNA has been developed using benzoylated, naphthoylated DEAE cellulose. All intracellular viral DNA is bound to a terminal protein. This protein is bound to the nascent strands synthesized in vitro by HU nuclear extracts as well.