PROTEIN-ASSOCIATED DNA BREAKS AS INDICATOR OF TOPOISOMERASE INHIBITION

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DNA topoisomerase I (top1) has become an essential target for anticancer research since the discovery that camptothecins are specific top1 poisons and exhibit promising anticancer activity. Camptothecins act by stabilizing top1-linked DNA breaks (cleavable complexes) which can be detected as protein-associated DNA breaks in drug-treated cells. Our studies are aimed at addressing several fundamental questions regarding the top1 cleavable complexes: (1) what are the molecular interactions between camptothecins and top1? (2) Can we discover and characterize new top1 inhibitors? (3) Are cleavable complexes trapped by other DNA alterations (base modifications, mismatches, DNA breaks, gaps...)? (4) How are the trapped cleavable complexes processed in cells and subcellular systems; and (3) what is the role(s) of topoisomerases in some cellular responses to DNA damage and apoptosis. Molecular interactions between camptothecins and top1 cleavable complexes have been addressed using an alkylating camptothecin derivative and a camptothecin- resistant point mutant top1. Both experimental approaches provide the most direct evidence that camptothecins block top1 by binding at the enzyme/DNA interface. Molecular modeling is consistent with this hypothesis. Two novel families of top1 inhibitors have been discovered in collaboration with outside groups: NSC 314622 by the COMPARE algorithm in the NCI Drug Screen, and aclarubicin by yeast screening. Cleavable complexes induce DNA damage after interference with replication and possibly transcription complexes. The resulting lesions are irreversible DNA breaks and top1 suicide complexes which are potentially recombinogenic. The processing (repair) of such lesions is yet unknown and is a goal of our studies. We are collaborating with Howard Nash (NIMH). In the absence of removal of cleavable complexes, top1 can kill cells (camptothecin-mimetic) or act as a DNA strand transferase and induce DNA recombinations. These effect are relevant to the cytotoxicity of camptothecins or other top1 poisons used in cancer treatment and to the cellular functions of top1.