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.
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