Our previously funded proposal was directed at testing the suitability of the purified human cell 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 our work was performed using three anticancer drugs with known structure-function activities; namely, camptothecin (CPT), ara-C and etoposide (VP16). The results of our studies strongly suggest that the DNA synthesome has the ability to interact with these agents as observed in intact cells. Therefore, in this application it is our intention to fully 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 by: (1) directly verifying that the in vitro interaction of the synthesome with anticancer agents known to affect DNA replication are representative of the molecular events occurring in the intact cell. This will be accomplished by analyzing the types of daughter DNA molecules produced by the complex, in DNA replication reactions performed in the presence and absence of these drugs using high resolution electrophoresis (HRE). The results will be compared with the DNA replication intermediates isolated from cells exposed to these same agents. (2) We will also perform detailed studies on the action of anticancer agents at the synthesome DNA replication fork by: (a) determining the effect of CPT, VP16 and ara-C and VP16 on their synthesome associated target-proteins; (c) determining the ability of ara-C to affect the coordinated synthesis of DNA by the synthesome; and (d) investigating the incorporation of ara-CMP by the synthesome into DNA at internucleotide linkages. (3) In addition, we propose studies to determine some of the biological consequences of internucleootide ara-CMP residues incorporated into DNA by the DNA synthesome.
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