Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins.

Abstract

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected [human cervical carcinoma] HeLa cells, was designated nuclear factor II. In the presence of Ad DNA with proteins at each 5'' end (Ad DNA-protein) and 3 proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol) and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase .alpha., .beta. and .gamma. activities contain a DNA topoisomerase activity. Type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. Evidently, a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.