STUDIES ON THE IN VITRO SYNTHESIS OF ADENOVIRUS DNA (ENZYMOLOGY, REPLICATION, ANTIBODIES, MOLECULAR CLONING, NUCLEAR FACTOR I)

Abstract

Data presented in this thesis demonstrate that a 3400 bp fragment of Ad DNA, mapping between coordinates 23.3 to 13.8, is expressed efficiently from a chimeric plasmid in E. coli. When inserted in the correct orientation downstream from transcription and translation initiation signals a 124 kDa polypeptide is formed. Antibody raised against this polypeptide strongly reacted with the 140 kDa DNA polymerase (Ad Pol) extracted from Ad-infected HeLa cells. Thus the data provides direct evidence that at least part of the Ad Pol is encoded by this fragment of viral DNA.;The Ad Pol and the 59 kDa Ad DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that when poly(dT)(.)oligo(dA) was used as a template(.)primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT)(.)oligo(dA). Two separate roles for the Ad DBP; one as a single strand DNA binding protein and one as a specific replication factor, are described. During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3' (--->) 5' exonuclease activity.;Nuclear Factor I (NF I) is a sequence specific DNA binding protein involved in the initiation and elongation reactions. Monoclonal antibodies have been used to demonstrate the ability of NF I to bind to the natural template, Ad DNA-prot. Protein-protein interactions have been examined using an indirect footprinting technique with Ad DNA-prot, and by using native polyacrylamide gels. These studies demonstrate complexes between NF I and DNA and suggest the presence of additional complexes involving pTP-Ad Pol and Ad DBP. Together, these studies have helped to explain the roles of the Ad Pol, pTP, Ad DBP, and NF I in catalysis of Ad DNA replication.