MUTATIONAL ANALYSIS OF E COLI DNA TOPOISOMERASE II

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DESCRIPTION: DNA topoisomerases are enzymes that alter the linking number of DNA and are responsible for the topological inter- and intraconversions that occur in DNA molecules. These conversions include the negative supercoiling of, the removal of negative supercoils from, the knotting of, and the catenation and decatenation of DNA. These enzyme are the known targets for antibiotics in bacterial cells and anti-tumor drugs in human cells. Topoisomerases can be classified into two distinct groups. Type 1 enzymes act by making a transient break in one strand of the DNA helix, passing a single or double strand DNA through the break, and resealing the break. Type 2 enzymes work in a similar manner except that they make a transient break in both strands of the helix. Examples of both types of enzymes exist in both eucaryotes and prokaryotes. DNA topoisomerase III (Topo III) is a type 1 enzyme that has been isolated from Escherichia coli. The crystal structure of the enzyme has been determined and is the only structure available of an active topoisomerase. This topoisomerase is unique among the topoisomerases in that it can act on both DNA and RNA substrates. In addition, homologues of this enzyme have been found to be encoded by promiscuous, broad host range plasmid genomes as well as the yeast and human genome. Thus far, E. coli is the only Topo III-like activity that is amenable to both biochemical and genetic analysis. The specific aims of this grant are: 1) assess the proposed model for type 1 topoisomerase-mediated relaxation and decatenation; 2) further identify the critical residues of the Topo III polypeptide involved in substrate binding and DNA cleavage using in vitro mutagenesis and attempt to elucidate the enzymatic mechanism at the molecular level; 3) attempt to identify cellular proteins that interact with Topo III and map the regions of Topo III with which they interact; 4) Suppress topB expression and analyze the effect on the expression of other topoisomerase genes and the state of the intracellular DNA.