It is now widely accepted that DNA replication occurs via a highly ordered series of concerted molecular reactions mediated by an organized complex of proteins which function together to faithfully copy genomic DNA. The applicant was the first to successfully isolate and purify a functional multiprotein DNA replication complex from human cells, which she designated the DNA synthesome. She has demonstrated that the DNA synthesome is fully competent to orchestrate, in vitro, all of the reactions required to efficiently and faithfully replicate DNA in vivo. Her previously funded application was directed at evaluating the DNA synthesome as an in vitro model system for defining the mechanism of action of anticancer drugs by comparing it to well studied cell-based model systems. Much of her work was performed using anticancer drugs with known structure-function activities. The results of her studies indicate that the DNA synthesome has the ability to interact, in many ways, with these agents as observed in intact cells. Therefore, in this application it is her intention to further develop, and demonstrate the utility of, the purified human cell DNA synthesome as an in vitro model system for investigating the action of anticancer drugs. A series of mechanistic studies are proposed to more closely define the action of camptothecin (CPT), ara-C and etoposide (VP16) on synthesome-mediated DNA replication. Each of the proposed studies grows from observation regarding the action of these drugs during the previous funding period.
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