The goal of this program is to determine how DNA topoisomerase-targeting therapeutics interact with their targets and what cellular processes are responsible for their efficacy. Yeast strains and topoisomerase mutants expressing cloned human topoisomerase I or II gene have been constructed for the screening of drugs that act on these enzymes, and for genetic and biochemical dissections of the mechanisms of cell-killing by these drugs. The dependence of drug cytotoxicity on the yeast homologs of the mammalian multidrug-resistance determinants, and on mutations or chemicals that interfere with cell-cycle control, will be examined to gain further insight on the mechanisms of topoisomerase-targeting drugs and to improve the sensitivity of the yeast system for drug screening. Direct experiments will be carried out to test whether transcription, in addition to replication, is involved in the cytotoxic action of amsacrine and etoposide type DNA topoisomerase II drugs. Expression of cloned human DNA TOP 2alpha and 2beta, which encode respectively the 170 and 180 kDa forms of DNA topoisomerase II, will be carried out in yeast to assess the relative importance of the two forms as drug targets. The existence of new DNA topoisomerases in human cells, particularly the homolog of the newly discovered yeast DNA topoisomerase III, will be examined for the purpose of identifying new targets of anticancer drugs. In addition, biochemical and structural studies will be done to obtain a detailed picture of drug-topoisomerase-DNA ternary complexes. The genetic, biochemical and structural studies proposed here aim at achieving a detailed molecular and atomic understanding of the drug-enzyme-DNA ternary complexes as well as rapidly applying the knowledge gained to pharmacology and clinical medicine.
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