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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM029006-16
Application #
2021864
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1981-04-01
Project End
1998-11-30
Budget Start
1996-12-01
Budget End
1997-11-30
Support Year
16
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
Chen, Yu-Tsung Shane; Wu, Jianhong; Modrich, Paul et al. (2016) The C-terminal 20 Amino Acids of Drosophila Topoisomerase 2 Are Required for Binding to a BRCA1 C Terminus (BRCT) Domain-containing Protein, Mus101, and Fidelity of DNA Segregation. J Biol Chem 291:13216-28
Chen, Stefanie Hartman; Plank, Jody L; Willcox, Smaranda et al. (2014) Top3? is required during the convergent migration step of double Holliday junction dissolution. PLoS One 9:e83582
Lee, Shun-Hsiao; Siaw, Grace Ee-Lu; Willcox, Smaranda et al. (2013) Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases. Proc Natl Acad Sci U S A 110:E3587-94
Chen, Stefanie Hartman; Plank, Jody L; Willcox, Smaranda et al. (2013) Improved methods for creating migratable Holliday junction substrates. Nucleic Acids Res 41:e60
Chen, Yu-Tsung; Collins, Tammy R L; Guan, Ziqiang et al. (2012) Probing conformational changes in human DNA topoisomerase II? by pulsed alkylation mass spectrometry. J Biol Chem 287:25660-8
Capp, Christopher; Qian, Yushen; Sage, Harvey et al. (2010) Separate and combined biochemical activities of the subunits of a naturally split reverse gyrase. J Biol Chem 285:39637-45
Wu, Jianhong; Feng, Liping; Hsieh, Tao-shih (2010) Drosophila topo IIIalpha is required for the maintenance of mitochondrial genome and male germ-line stem cells. Proc Natl Acad Sci U S A 107:6228-33
Wu, Jianhong; Phatnani, Hemali P; Hsieh, Tao-Shih et al. (2010) The phosphoCTD-interacting domain of Topoisomerase I. Biochem Biophys Res Commun 397:117-9
Capp, Christopher; Wu, Jianhong; Hsieh, Tao-Shih (2010) RecQ4: the second replicative helicase? Crit Rev Biochem Mol Biol 45:233-42
Collins, Tammy R L; Hsieh, Tao-Shih (2009) Monitoring the topoisomerase II DNA gate conformational change with fluorescence resonance energy transfer. Methods Mol Biol 582:59-70

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