The topology of DNA has a profound influence on how its genetic information is regenerated, rearranged, and expressed in vivo. Consequently, the enzymes that regulate the topological structure of nucleic acids play a crucial role in controlling the cellular function of DNA. One class of enzymes, the type II topoisomerases, catalyze changes in the topological state of DNA by passing one intact DNA helix through a transient double- stranded break made in a second helix. These ubiquitous enzymes exist in vivo as phosphoproteins, are essential for the viability of eukaryotic cells, and are involved in many aspects of DNA replication, transcription, repair, chromosome structure, nuclear structure, and chromosome segregation. In addition, topoisomerase II appears to be the primary cellular target for several major classes of antineoplastic agents. Despite the importance of topoisomerase II to the eukaryotic cell and to the clinical treatment of human cancers, very little of its enzymatic mechanism or physiological regulation is understood. Therefore, the ultimate goal of this proposal is to define the function and biology of the enzyme in eukaryotic cells. More specifically, the aims of this proposal are 1) to describe the mechanism of the topoisomerase II-catalyzed double-stranded DNA passage reaction, 2) to determine the role of serine phosphorylation as a physiological regulator of the enzyme, and 3) to determine the mechanism by which anti-neoplastic agents alter the activity of topoisomerase II. Drosophila Kc tissue culture cells, an established embryonic line, will be employed as a research model. In order to more fully characterize the double-stranded DNA passage reaction of topoisomerase II, a kinetic approach will be coupled with enzyme- substrate binding studies, chemical modification experiments, and a conformational analysis of the structural transitions undergone by the enzyme during the course of DNA strand passage. The role of serine phosphorylation as a physiological regulator of the enzyme will be assessed by examining the kinetic properties of modified topoisomerase II, defining in vivo and in vitro sites of phosphorylation, identifying the enzymes responsible for the in vivo phosphorylation/dephosphorylation of topoisomerase II, and by correlating levels of modification with the cellular activity and location of the enzyme. Topoisomerase II-antineoplastic drug interactions will be studied by kinetic and binding experiments, by generating mutant cell lines with drug-resistant enzymes, and by defining relationships between enzyme concentration in vivo and drug-induced cytotoxicity.
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