The long-term goal is to elucidate the biochemical, molecular and cell biological events involved in eukaryotic DNA replication, and in so doing, to identify rational approaches to the specific inhibition of DNA replication in vivo. It is anticipated that the specific inhibition of DNA synthesis will represent a viable means to approach the pharmacologic control, both of malignant cellular proliferation and in vivo viral replication. During the initial phase, work will be largely restricted to higher eukaryotes with emphasis on human KB cells and Drosophila melanogaster embryos as experimental model systems. The application of mechanism-based inhibitors of human DNA polymerase Alpha as general purpose affinity ligands for the purification of DNA replication proteins will be investigated. Using these ligands, DNA polymerase Alpha holoenzyme will be purified from Drosophila embryos and compared with the conventionally purified polymerase. Individual polypeptide subunits will be separately purified following mild denaturation of the holoenzyme. Antibodies will be prepared against each of the holoenzyme subunits. In vitro enzymologic studies of Drosophila DNA polymerase Alpha holoenzyme will be performed in order to characterize the molecular mechanism of substrate recognition and in vitro DNA synthesis. This mechanism will be compared with that previously determined for the human enzyme. Mechanism-based probes will be developed for UV-photo-affinity crosslinking analysis of the active site(s) of polymerase catalysis. Reconstitution of the enzymatic activities of DNA polymerase Alpha holoenzyme from separated polypeptide subunits will also be attempted, as will the use of specific antibodies to study the roles of individual subunits in the mechanism of DNA synthesis. The physical interaction between DNA polymerase Alpha and its various macromolecular substrates will be examined directly using scanning transmission electron microscopy. Immunocytochemical studies will be performed in order to determine the intracellular and intraorganismal localization of DNA polymerase Alpha as a function both of development and differentiation. In vivo studies will focus on the development of """"""""single-cell"""""""" assay systems to study DNA replication, and specifically, the microinjection of mechanism-based inhibitors of DNA polymerase Alpha to determine the potential of these molecules to block DNA synthesis in vivo. It will also be possible to microinject specific antibodies directed against other nuclear proteins to assess their role(s) in vivo.
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