ENZYMATIC AND TEMPLATE REQUIREMENTS FOR THE IN VITRO SYNTHESIS OF ADENOVIRUS DNA (REPLICATION, ORIGIN, ADENOVIRUS, HOST FACTORS)
GUGGENHEIMER, RONALD A.
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Two proteins required for the in vitro synthesis of full-length Adenovirus DNA have been purified from nuclear extracts of uninfected Hela cells and have been designated nuclear factors I and II. Purifiction of both nuclear factors was aided by the use of an in vitro biochemical complementation assay in which Adenovirus DNA-protein complex (Ad DNA-prot) was used as the template.;Initiation of Ad DNA replication proceeds via the synthesis of a covalent complex between the 80 kDa pTP and 5' dCMP, the first nucleotide of the nascent DNA strand. Using Ad DNA-prot as the template, the covalent addition of dCMP to the pTP requires the Ad Pol, as well as the pTP, and, in the presence of the Ad DBP, is stimulated as much as ten-fold by nuclear factor I. Elongation of the pTP-dCMP initiation complex, using the 3' OH of the dCMP residue as a primer terminus can occur in vitro. In the presence of Ad DNA-prot, ATP, Mg('2+), the 4 dNTPs, the pTP, Ad DBP and nuclear factor I, the Ad Pol can synthesize DNA chains 25-35% the length of Adenovirus DNA. Omission of nuclear factor I reduces the rate and extent of DNA synthesis. Addition of nuclear factor II leads to the synthesis of full-length Ad DNA at a rate that approximates the observed rate of Ad DNA synthesis in vivo.;Nuclear factor I has been shown to be a site specific binding protein. DNase I "footprinting" experiments have localized the nuclear factor I binding site to a region between nucleotides 17-48 of the Ad genome, close to the origin of Ad DNA replication. Nuclear factor II has been shown to be a type I DNA topoisomerase. Type I DNA toposiomerases isolated from Hela cells and calf thymus can substitute for the nuclear factor II complementing activity in the in vitro Ad DNA replication system, while the E. coli type I topoisomerase (protein) does not substitute for nuclear factor II.;Studies in which the nucleotide sequence requirements for Ad DNA replication were determined utilized a plasmid DNA (pLA1) containing the replication origin of Ad DNA lacking the 55,000 dalton terminal protein. Replication of plasmid pLA1 DNA was initiated by a protein-primed mechanism in a manner similar to that found with Ad DNA-prot and required (i) that the cloned Adenovirus ori region be present at the terminus of a linearized (form III) DNA molecule and (ii) the pTP, Ad DBP, Ad Pol, nuclear factor I and an additional host protein, designated factor pL. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI.