Although the genetic information of DNA is embodied in its primary structure, the topological state of the nucleic acid molecule has a profound influence on its physiological function. In vivo, the topological structure of DNA is modulated by a ubiquitous class of enzymes known as DNA topoisomerases. In prokaryotes, these enzymes affect such fundamental processes as DNA replication, recombination, repair, and transcription. Unfortunately, the cellular functions of eukaryotic topoisomerases are not yet clear. Therefore, the ultimate goal of this project is to define the role of DNA topoisomerases in eukaryotic organisms. To this end, the function and biology of the type II topoisomerase from the fly, Drosophila melanogaster, will be determined. More specifically, the aims of this proposal are 1) to describe in detail the interactions between Drosophila topoisomerase II and its substrates, DNA and ATP; 2) to isolate and characterize the gene which codes for the enzyme; 3) to determine how the activity of topoisomerase II is regulated in D. melanogaster; and 4) to define the in vivo function of the enzyme. This research is designed to take advantage of the known physicochemical properties of the enzyme and the biological attributes of Drosophila. The enzymology of topoisomerase II will be characterized by a variety of biochemical and physical techniques, such as kinetic studies, binding assays, chemical modification, electron microscopy, and fluorescence spectroscopy. The regulation and cellular function of the enzyme will be elucidated by classical genetic and biological techniques coupled with recombination DNA methodologies. Procedures to be employed include genetic crosses, isolation of mutant flies, in situ hybridization, molecular cloning, DNA sequencing, and transformation on Drosophila with mobile genetic elements. Since topoisomerases affect several aspects of DNA metabolism, this study may illuminate the events which underlie those congenetal birth defects resulting from gross chromosomal abnormalities. Moreover, the topological structure of DNA is altered in cancerous cells and DNA topology and/or topoisomerase activity is important for the transformation of host genomes by viruses and mobile genetic elements. Therefore, this work may provide insight into some of the mechanisms of cancer.
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