THE HUMAN CELL MULTIPROTEIN DNA-REPLICATION COMPLEX (MRC) - THE EFFECT OF CAMPTOTHECIN ON ITS ABILITY TO SUPPORT IN-VITRO DNA-SYNTHESIS

Purpose : We have previously reported on the isolation and characteriz ation of a multiprotein DNA replication complex (MRC) from HeLa cells that fully supports in vitro DNA replication. Based upon its ability t o replicate DNA in a cell-free environment (devoid of other cellular p rocesses) the MRC may serve as a unique model system for investigating the mechanisms of action of anticancer drugs that directly affect DNA synthesis. The experiments described in this report were performed to establish whether the MRC could serve as a model system to examine in detail the mechanism of action of camptothecin, a DNA topoisomerase I inhibitor, Methods: We examined the effects of increasing concentrati ons of camptothecin on HeLa cell survival, intact HeLa cell DNA synthe sis and MRC-mediated in vitro DNA replication. We also performed topoi somerase I assays in the presence of increasing concentrations of camp tothecin to study the direct effects of the agent on MRC-associated to poisomerase I activity. Furthermore, we employed an SDS precipitation assay to measure the formation of MRC-associated topoisomerase I-cleav able complexes in the presence of increasing concentrations of camptot hecin. Results: We found a close correlation between the IC50 values f or intact HeLa cell DNA synthesis (0.15 mu M) and MRC-mediated in vitr o DNA synthesis (0.05 mu M). Similarly, we found that 0.05 mu M campto thecin inhibited MRC-associated topoisomerase I activity by approximat ely 50%. In addition, we found that the formation of MRC-associated to poisomerase I-cleavable complexes increased linearly with increasing c oncentrations of camptothecin. Conclusions: The data presented in this report support the use of the MRC as a model system to study the mech anism of action of camptothecin. We anticipate that future studies wit h the MRC will help elucidate the cellular consequences of camptotheci n-cleavable complex formation.