The long range goal of these studies is to described, at the molecular level, DNA replication and its regulation in mammalian cells. A cell free replication system capable of replicating exogenous DNA templates with a viral origin of replication has been developed in this laboratory, allowing the mammalian proteins involved in DNA replication to be identified by biochemical methods such as fractionation and reconstitution experiments. DNA helicases are known to play a central role in the replication of prokaryotic and viral genomes, and can therefor be expected to be similarly important in mammalian replication. We will attempt to purify and characterize the cellular helicase(s) involved in replication using the resources and technical expertise acquired by this laboratory in the investigation of our cell free replication system. The initial fractionation steps will use the chromatographic columns that proved useful in isolating the replication proteins identified so far. Subsequent steps will include affinity columns such as DNA-cellulose and ATP-agarose and will be based in part on published experience with the purification of helicases from calf thymus and murine FM3A cells. The role of helicases in SV40 replication will be defined by assaying for reconstitution of the replication reaction with purified proteins. Their mechanism of action will be investigated by defining substrate specificity and interactions with other purified components of the replication reaction in s variety of assays, such as origin dependent unwinding, reduction of time lag of initiation, influence on kinetics and processivity of elongation, polarity of translocation, and modulation of topoisomeraseinduced changes in linking number. Monoclonal antibodies will be raised, allowing immunoaffinity-chromatography to facilitate purification. They will allow investigation of cell cycle regulation and subcellular localization as indirect means of defining the role of cellular helicases. Given a sufficient amount of pure protein, protein sequencing and cloning of a helicase gene will be attempted. The elucidation of the mechanisms of replication in mammalian cells will lead to a better understanding of its regulation and provide insight into the fundamental alterations responsible for generation and maintenance of neoplasia. This may also provide the tools and targets necessary to design new and more specific antineoplastic agents.
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