The overall objective of this proposal is to unravel the molecular mechanism by which adenovirus DNA replicates in animal cells. Adenovirus DNA replication system is an excellent model system to understand how other viruses replicate in the infected host cell and in addition, how DNA replication initiates in a eukaryotic organism, in general. Two viral coded gene products, the precusor of terminal protein, which is covalently attached to the mature viral DNA (pTP) and a DNA polymerase (Ad Pol) are the essential components of viral DNA replication in vitro and in vivo. Purification of the proteins from the adenovirus-infected HeLa cells yielded a stable complex which is essential for initiation and elongation of DNA replication. A transient expression system in monkey cells, achieved by DNA-mediated transfection methodology has provided preliminary evidence that 1) there is a nuclear targeting signal domain in pTP, which targets this protein to the nucleus of the transfected cells, and 2) both pTP and Ad Pol interact, when their respective genes were co-transfected. In order to understand the molecular mechanisms of DNA replication, it is important to identify the functional domains of pTP and Ad Pol involved in this interaction. A large number of mutants will be isolated in the coding sequences of pTP and Ad Pol by 1) linker-insertion/deletion 2) linker scanning 3) deletion loop and 4) mixed oligonucleotide mutagenesis methods. These mutants will be assayed, using immunofluorescence microscopy, immunoprecipitation techniques and by in vitro replication reactions, to identify the signal domain of pTP involved in its nuclear localization and the domains of pTP and Ad Pol involved in interaction for their function in the initiation of DNA replication. The proposed model that the homologous adenovirus DNA binding protein interacts with Ad Pol during DNA replication will also be tested by using these methods.
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