STUDIES ON THE MECHANISM OF ADENOVIRUS DNA REPLICATION

Abstract

The mechanism of replication of the 35 kilobase pair adenovirus genome was investigated. An in vitro DNA replication system was developed and characterized which faithfully mimicked in vivo replicaton in that, (1) the reaction required viral DNA with intact 55K terminal proteins (Ad DNA-pro), (2) replication initiated specifically at either end of the template and proceeded by continuous elongation with displacement of the parental DNA strand, (3) full length progeny Ad DNA-pro was the major product of the reaction, and (4) mutants thermolabile for DNA replication in vivo gave rise to extracts that were thermolabile in vitro. The in vitro replication reaction required ATP, MgCl(,2), 4 deoxynucleoside triphosphates, Ad DNA-pro, and two cell extracts. One extract was a preparation of cytosol from infected HeLa cells; cytosol from uninfected cells was inactive. The second was a nuclear extract which was active when prepared from either infected or uninfected cells.;Initial analysis of this system provided evidence that the 80K precursor (pTP) of the 55K terminal protein serves as the primer for replication. The first nucleotide in the nascent chain is dCMP. In the presence of (alpha)P('32) dCTP, the pTP present in all the cytoplasmic extract was shown to form the product pTP-dCMP. None of the other deoxynucleotides substituted for dCTP in this reaction. With the addition of dTTP, dATP and ddGTP to the reaction mixture, the product elongated 26 nucleotides to the first dGMP in the nascent chain, forming a pTP-linked 26mer. These results showed that the pTP specifically binds dCMP and that pTP-dCMP can function as an intermediate in the in vitro initiation reaction.;The requirements for the early steps in replication were further studied by purification of proteins form the crude extracts. Using pTP-dCMP synthesis as a specific assay for the pTP, and a DNA synthesis complementation assay to detect other components of the replication machinery, it was found that the cytoplasmic extract could be fully replaced by the virus coded DNA binding protein (Ad DBP) and a second fraction, which contained the 80K pTP in an equimolar complex with a 140K protein. In the course of the purification a new DNA polymerase (Ad Pol), distinguished both in its chromatographic and enzymatic properties from all known host polymerases, was discovered. The Ad Pol was shown to be associated with the 140K protein that copurified with the pTP. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI.