The long term objective of our proposed research is studying the structure and function of chromosomes. Our efforts currently focus on the function and mechanism of a type II DNA topoisomerase from Drosophila melanogaster. Since DNA topoisomerase can alter the overall structures of chromosomes, they take part in regulating chromosome functions. We have purified the type II topoisomerase from Drosophila embryos and prepared specific antibodies against this enzyme. These antibodies will be used as a reagent in many of our experiments. We will first examine structural evolution of eucaryotic DNA topoisomerases by monitoring the binding of antibodies to these enzymes. We will also examine the state of in vivo chemical modification of Drosophila topoitomerase and the effects of the chemical modification on enzymatic activities. Phosphorylation and poly ADP-ribosylation will be analyzed first since they both may have profound effects on the topoisomerase activities. We will also continue our investigation of the double-strand DNA cleavage reaction by DNA topoisomerase II. The stimulating effects of several DNA intercalating drugs on this reaction will be studied first. We will analyze the sequence specificity in the cleavage reaction, especially for the strong cleavage sites in the cloned Drosophila DNA sequence. For the functional studies of this enzyme, we will first characterize the proteins specifically associated with DNA topoisomerase in the chromatins. We will use immunochemical methods to examine if these proteins are part of DNA topoisomerase I, DNA and RNA polymerases. To establish a groundwork for our future experiments in using genetics to probe the functions of this enzyme, we will pursue the molecular cloning of the DNA sequence encoding this enzyme. We will use an inducible expression vector for our cDNA cloning experiment and the transformants will be screened with an immunochemical method. The structure, expression and the chromosome location of this gene will be studied.
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