There currently exists a critical need for improved model systems which will rapidly decipher the mechanism of action of clinically useful anticancer agents. this proposal describes experiments which utilize a complex of proteins which have been recently isolated from human cells, and which have been demonstrated to efficiently support the replication of DNA in vitro. These experiments will test whether this complex of DNA replication proteins can interact with each of three clinically useful anticancer drugs, camptothecin, VP-16 and ara-C (which are known to directly affect DNA synthesis), and mimic the known effects of these drugs on DNA replication.
Our aims are to: (1) validate the suitability of the human cell DNA replication complex for defining the mechanism of action affected by these anticancer drugs by comparing the complex model system to other cell based model systems. To do this, we will confirm that the concentrations of these drugs found to inhibit intact cell DNA synthesis and cell proliferation also interfere with purified multiprotein complex mediated DNA replication. direct verification that the in vitro interactions of these drugs with the purified multiprotein complex are representative of the molecular events occurring in the intact cell will be made by comparing the results of high resolution electrophoretic agarose gel analyses of the multiprotein complex's DNA replication intermediates produced in the presence of these drugs, with those replication intermediates generated in drug exposed cells. (2) Initiate studies to demonstrate the usefulness of the purified multiprotein complex model system for investigating the mechanism of drug action. We will determine the effect of these drugs on the kinetics of the multiprotein complex's DNA replication initiation, elongation and termination reactions by using a Southern blot based assay we have developed to precisely determine when the multiprotein complex initiates DNA synthesis, how long the complex takes to successfully elongate the replicating DNA, and when it completes (terminates) a daughter DNA molecule. If the Southern blot analyses indicate that the DNA replication products formed in the presence of these drugs are halted at specific DNA sequences, then experiments will be performed to precisely map these DNA sequences. Also, the effect of these drugs on their multiprotein complex associated """"""""target-proteins"""""""" will be determined, and correlations made with these drugs' interactions using purified enzymes. This project has the immediate potential for establishing the purified human cell multiprotein complex as a unique model system for advancing our understanding of how anticancer drugs, affecting DNA synthesis, actually function. The long term objective of our collaborative research efforts is to develop a clearer understanding of action of these anticancer drugs, and the role mechanisms regulating DNA synthesis play in controlling cell proliferation.
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