The topological state of DNA in the cell is regulated by ubiquitous enzymes known as topoisomerases. The type II enzyme alters nucleic acid topology by passing a DNA helix through a transient double-stranded break that it creates in the backbone of a separate helix Topoisomerase II is an essential enzyme that exists in the cell as a phosphoprotein. It is required for chromosome segregation and organization and is involved in a number of fundamental DNA processes including replication, transcription, and recombination. Beyond its physiological functions, topoisomerase II is the primary cellular target for some of the most potent drugs currently employed for the treatment of human cancers. The clinical efficacies of these agents correlate with the ability to stabilize covalent topoisomerase II-cleaved DNA complexes that are intermediates in the enzyme's catalytic cycle. Despite the importance of topoisomerase II to the eukaryotic cell and to the chemotherapeutic treatment of cancer, relatively little is understood concerning the enzyme's mechanism of action or its physiological regulation. Therefore, the ultimate goal of the proposed research is to define the function and biology of topoisomerase II. More specifically, the aims of this proposal are 1) to further define the catalytic mechanism of topoisomerase II, 2) to characterize the regulation of the enzyme by phosphorylation, and 3) to delineate the mechanism by which topoisomerase II-targeted drugs alter the enzyme's activity. Drosophila and yeast will serve as the research models for the proposed studies. Drosophila topoisomerase II is by far the most well characterized eukaryotic topoisomerase and yeast allows a degree of genetic manipulation that is unmatched by any other eukaryotic organism The catalytic mechanism of topoisomerase II will be further defined by a combination of kinetic and physical studies. Experiments will analyze both the wild type and mutant forms of the enzyme and will take great advantage of novel assays developed during the previous grant period. The role of phosphorylation as a physiological regulator of the type Ill enzyme will be assessed by further characterizing the effects of modification on the enzyme's catalytic cycle, mapping phosphorylated amino acid residues, and determining the in vivo effects of deleting topoisomerase II phosphorylation sites. Finally, the mechanism of action of topoisomerase II targeted antineoplastic drugs will be delineated by analyzing the effects of drugs on DNA cleavage/religation at specific enzyme recognition sites, defining topoisomerase II-drug interactions, and analyzing the properties of drug-resistant type II enzymes,
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