42] Adenovirus DNA replication in a reconstituted system

Abstract

Publisher Summary This chapter describes adenovirus (Ad) DNA replication in a reconstituted system. Reconstitution of Ad DNA replication using purified proteins leads to efficient initiation followed by rapid elongation at rates comparable to those measured in infected cells. In vitro initiation requires a short lag period and is linear for at least 30 min, indicating that possibly other factors are involved in this rate-limiting process. In vivo replication occurs in localized foci containing presumably high concentrations of replication proteins and that DNA is attached to the nuclear matrix through the terminal protein. Crude nuclear extracts of HeLa cells infected with adenovirus type 5 (Ad5) was the first system in higher eukaryotes for which such an assay became possible, presumably due to the high level of replication proteins present in these nuclei. Subsequent studies led to the characterization of the mechanism of DNA replication, which occurs by protein priming, and the purification of the various replication proteins. Based on, among others, mutant complementation, three viral proteins are required, all encoded by one early transcription unit (E2). These are the viral DNA-binding protein (DBP), the precursor terminal protein (pTP), and the DNA polymerase (Pol).