The long term goal of the proposed research is the analysis of structure and function of eucaryotic chromosome. The current research activity is focused on the structure, function, and mechanism of DNA topoisomerases, a family of enzymes which play a critical role in regulating the structure/function of DNA and chromosomes. DNA topoisomerases can mediate the DNA structural transition by reversibly breaking DNA backbone bonds and passing DNA strands through these transient DNA breaks. The interest in DNA topoisomerases is also heightened by the recent discovery that they are the intracellular pharmacological targets of a number of clinically important anti-tumor and antibiotic drugs. Therefore, furthering our understanding of the molecular mechanism and biological function of DNA topoisomerases is of immediate relevance to the development of anti-topoisomerase drugs as chemotherapeutic agents.
The specific aim of the proposed research will encompass two major areas: structure/function analysis of DNA topoisomerases and the study of biological functions of these enzymes. The structural and functional analysis of DNA topoisomerases are facilitated by efficient heterologous expression system for both type I and II enzymes from Drosophila melanogaster. For the type II DNA topoisomerase, region-specific mutagenesis will be carried out in a domain which is possibly involved in coordinating the ATPase function and DNA breakage/rejoining activity of this enzyme. Another region of interest in topoisomerase II is the nonhomologous, hydrophilic C-terminus which may have a unique in vivo function. The availability of linker-insertional mutants generated in this laboratory will provide a handle to engineer the protein for further biochemical analysis. For type I DNA topoisomerase, we will focus on the functional analysis of its hydrophilic amino terminus, using molecular genetic approaches. The biological functions of DNA topoisomerase will be studied by a molecular genetic approach. There are a number of new topoisomerase I mutants generated in our laboratory and they can provide us valuable tools in defining the critical physiological role of DNA topoisomerase I during the growth and development of a multi-cellular organism.
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