Camptothecin (CPT) and its analogs are topoisomerase I (Topo I) inhibitors and show efficacy against solid tumors which have been historically resistant to most cancer chemotherapeutic agents. The long-term objective of the present application is to elucidate the molecular mechanism of DNA replication inhibition in CPT-treated cells. The cytotoxicity of CPT is mainly exerted on S-phase cells and is associated with an inhibition of DNA replication. This inhibition of DNA replication is thought to be mediated by cis-acting processes as a result of the collision of the advancing replication fork with the cleavable CPT-Topo I-DNA complex. Work carried out in this laboratory suggests that in addition to cis-acting inhibitory processes, there are also trans-acting inhibitory processes active in CPT-treated cells. The investigator hypothesizes that damaged/modified DNA produced by CPT is a mediator that recruits RPA for repair and inhibits its function in DNA replication. It is further hypothesized that Ku may either compete with RPA for binding to damaged/modified DNA, or disassemble or remodel the RPA-DNA complex to release RPA. Thus RPA is allowed to initiate DNA replication again, and therefore the inhibition is reduced. Information on the molecular determinants of this regulation of DNA replication may offer new ways of intervention in the clinic for achieving CPT sensitization.
The Specific Aims focus on studying RPA and Ku as the activities responsible for the regulation of DNA replication in CPT-treated cells in the framework of the above model. The goals are: 1. To characterize modifications in the properties of RPA that are related to DNA replication inhibition in CPT-treated cells and 2. To study how Ku affects DNA replication inhibition in CPT-treated cells, and by what mechanism it interacts and modulates the activity of RPA. The Simian virus 40 (SV40) based in vitro DNA replication assay will be used in the proposed experiments. The proposed research is based on information from preliminary results and combines current knowledge on the DNA replication and CPT cytotoxicity to characterize the modulation of DNA replication in CPT-treated cells.
