The DNA topoisomerases, a family of ubiquitous enzymes that participate in nearly all cellular processes involving DNA, catalyze the passage of DNA strands or double helices through one another. The long range goal of this project has been the elucidation of the reaction mechanisms and cellular functions of these enzymes. Mechanistic studies proposed in this application focus on the type II enzymes that transport one DNA double helix through another. A combination of mutagenesis and biochemical experiments will be carried out to test and further refine a molecular model in which large and specific interdomainal movements, triggered by ATP binding to a DNA-bound enzyme, are involved in various reaction steps. The proposed functional studies of these enzymes focus on testing three specific postulates: (1) that a DNA-bound type II topoisomerase may make the DNA more prone to breakage under mechanical tension during mitosis; (2) that mammalian DNA topoisomerase II beta, one of the type II topoisomerase isoforms, may have a critical role in neural development; (3) an interacting pair of enzymes, a DNA helicase and a type IA DNA topoisomerase that catalyzes the passage of one DNA single-strand through another, may be specifically involved in preventing R-loop formation between a nascent RNA and its DNA template. Additional studies are planned to address the molecular roles of the various enzymes involved in these processes. Experiments are also proposed to further test a fourth hypothesis that at least in bacteria the formation of a cellular patch of a DNA binding protein, through protein-mediated subcellular localization, may lead to the silencing of genes linked to but well-separated from the recognition sequence of the particular DNA binding protein. The proposed studies address a number of issues fundamental to cellular processes involving DNA, and these issues are directly related to problems of key medical importance, in particular the maintenance of genome stability in somatic cells, including well-differentiated neurons with a long life span.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM024544-27
Application #
6603741
Study Section
Biochemistry Study Section (BIO)
Program Officer
Lewis, Catherine D
Project Start
1977-07-01
Project End
2005-06-30
Budget Start
2003-07-01
Budget End
2005-06-30
Support Year
27
Fiscal Year
2003
Total Cost
$571,942
Indirect Cost
Name
Harvard University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
082359691
City
Cambridge
State
MA
Country
United States
Zip Code
02138
Azarova, Anna M; Lyu, Yi Lisa; Lin, Chao-Po et al. (2007) Roles of DNA topoisomerase II isozymes in chemotherapy and secondary malignancies. Proc Natl Acad Sci U S A 104:11014-9
Kwan, Kelvin Y; Greenwald, Rebecca J; Mohanty, Subhasis et al. (2007) Development of autoimmunity in mice lacking DNA topoisomerase 3beta. Proc Natl Acad Sci U S A 104:9242-7
Lyu, Yi Lisa; Lin, Chao-Po; Azarova, Anna M et al. (2006) Role of topoisomerase IIbeta in the expression of developmentally regulated genes. Mol Cell Biol 26:7929-41
Stupina, Vera A; Wang, James C (2005) Viability of Escherichia coli topA mutants lacking DNA topoisomerase I. J Biol Chem 280:355-60
Jiang, Xiaofeng (2005) Random mutagenesis of the B'A' core domain of yeast DNA topoisomerase II and large-scale screens of mutants resistant to the anticancer drug etoposide. Biochem Biophys Res Commun 327:597-603
Stupina, Vera A; Wang, James C (2004) DNA axial rotation and the merge of oppositely supercoiled DNA domains in Escherichia coli: effects of DNA bends. Proc Natl Acad Sci U S A 101:8608-13
Lyu, Yi Lisa; Wang, James C (2003) Aberrant lamination in the cerebral cortex of mouse embryos lacking DNA topoisomerase IIbeta. Proc Natl Acad Sci U S A 100:7123-8
Kwan, Kelvin Y; Moens, Peter B; Wang, James C (2003) Infertility and aneuploidy in mice lacking a type IA DNA topoisomerase III beta. Proc Natl Acad Sci U S A 100:2526-31
Wang, Yong; Lynch, A Simon; Chen, Sue-Jane et al. (2002) On the molecular basis of the thermal sensitivity of an Escherichia coli topA mutant. J Biol Chem 277:1203-9
Wang, Yong; Lyu, Yi Lisa; Wang, James C (2002) Dual localization of human DNA topoisomerase IIIalpha to mitochondria and nucleus. Proc Natl Acad Sci U S A 99:12114-9

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