The long term goal of this research proposal is to analyze the structure and function of eucaryotic chromosomes. The focus here is on the functional and mechanistic analysis of a eucaryotic DNA topoisomerase II. Since DNA topoisomerase can alter the structure and topology of DNA molecules. they play important roles in modulating the functions of DNA and chromosomes. Furthermore, since DNA topoisomerases are shown to be the intracellular targets for a number of clinically imPortant antibiotics and anti.tumor agents. A structure and function analysis of these enzymes can help elucidate the mechanism of action of these potent pharmacologic agents. The gene encoding Drosophila DNA topoisomerase II has been isolated, sequenced and over-produced in the yeast expression system. This heterologous yeast expression system will be used to facilitate the structural and functional analysis of the Drosophila enzyme. Site/region specific mutations will be introduced into certain domains, which we believe might be functionally important, and their biochemical properties will be investigated using the yeast expression system for isolating the mutant enzymes in quantity. The biological functions of these mutant forms can be first tested with the genetic complementation in yeast since the wildtype Drosophila enzyme can complement the null and temperature sensitive mutations of yeast TOP2 gene. The mutant TOP2 will also be reintroduced into fly by the germ line transformation and its function during the development of Drosophila can be analyzed. We have mapped the cytogenetic location of Drosophila TOP2 and attempts have been initiated to identify if any known lethal in this cytogenetic region correspond to a deficiency in TOP2. The identification of Top2 lethal can enable us to test the physiological functions of TOP2. The mechanistic studies of topoisomerase II will be focused on two aspects: protein/DNA interactions and characterization of reaction intermediates. We will also establish a biochemical assay with which we can analyze the inter-molecular strand transfer reaction mediated by topoisomerase II. The strand transfer reaction could be of significance in addressing the possible role of topoisomerase II in chromosome rearrangement.
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