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 campothecins 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) How are the trapped cleavable 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. Camptothecin derivatives modified at other positions will be used to test our molecular modeling. 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 have also shown that top1 can be trapped by damaged DNA using oligonucleotides containing base mismatches, abasic sites, and various types of breaks. Under these conditions, top1 can act as a DNA strand transferase and therefore can induce DNA recombinations. This effect is relevant to the cytotoxicity of camptothecins and to the cellular functions of top1.