Topoisomerase II is an essential enzyme that is required for proper chromosome structure and segregation and plays important roles in DNA replication and recombination. Beyond its critical cellular functions, it is the primary target for some of the most active and widely prescribed drugs used for the treatment of human cancers. These agents elicit their cytotoxic effects by a mechanism that is markedly different than that of other drugs. Rather than inhibiting the catalytic activity of topoisomerase II, anticancer drugs targeted to the enzyme dramatically increase levels of covalent topoisomerase II-cleaved DNA complexes that are normal, but fleeting, catalytic intermediates. When the resulting topoisomerase II-associated double-stranded DNA breaks are present in high concentrations, they generate mutations, chromosomal translocations, and trigger cell death pathways. Although topoisomerase II is one of the most important targets for cancer chemotherapy, there is compelling circumstantial evidence that the enzyme also has the potential to trigger the disease. Indeed, secondary leukemias associated with specific chromosomal translocations are observed in some patients treated with topoisomerase II-targeted drugs. Because topoisomerase II must create double-stranded breaks in DNA in order to function, the enzyme poses an intrinsic threat to genomic integrity every time it acts on the genetic material. Despite the central importance of topoisomerase II to the cancer problem, interactions of the enzyme with DNA and anticancer drugs have not been well characterized. Thus, the ultimate goal of this proposal is to further delineate the mechanism by which topoisomerase II carries out its fundamental cellular reactions and the mechanism by which drugs alter the catalytic function of the enzyme. Research models for this study will be human, Paramecium bursaria Chlorella virus-1 (PBCV-1), yeast (Saccharomyces cerevisiae), and Drosophila.
The specific aims of this proposal are to: 1) further define the catalytic mechanism of topoisomerase II; 2) determine the basis for the exceptionally robust DNA cleavage activity of PBCV-1 topoisomerase II; 3) further delineate the mechanistic basis for the actions of topoisomerase II-targeted anticancer drugs; and 4) explore relationships between topoismerase II and genomic stability. The proposed experiments are based on a number of novel findings made during the previous grant cycle, including the discovery of PBCV-1 topoisomerase II, the first eukaryotic viral type II enzyme to be characterized. Studies will take great advantage of several assays that were developed in the principal investigator's laboratory and will utilize biochemical, physical, and genetic approaches to address the stated aims of the proposal.
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