ROLE OF TOPOISOMERASES IN DNA METABOLISM

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It is the goal of this project to provide a biochemical basis for the role of topoisomerases in DNA metabolism. These enzymes are clearly implicated in the management of DNA strands during DNA replication, transcription, and recombination. In prokaryotes, topoisomerases are the target of clinically significant antibiotics, while in eukaryotes they are the target of promising antineoplastic agents. Thus a full understanding of their mode of action should provide a sounder basis for rational drug therapy. Topoisomerase function is investigated by modeling their action in vitro using systems reconstituted with purified proteins. One central point of topoisomerase action is during the final stages of DNA replication, when DNA synthesis must cease and the daughter chromosomes must be untangled. Using systems where small plasmid DNAs undergo either unidirectional or bidirectional replication, we will study the influence of the three Escherichia coli topoisomerases, DNA gyrase, topoisomerase I (Topo I), and topoisomerase III (Topo III) on the termination and segregation stages of DNA replication. The disposition after termination of the replication fork components will be determined and the mechanism of action of Tus protein-catalyzed arrest of replication fork progression elucidated. This protein, when complexed to specific terminator sequences, causes replication termination during E. coli chromosomal DNA replication. A more complete picture of the final stages of the replication process should provide useful information for our developing understanding of the events necessary for the sorting of chromosomes and the initiation of cell division. The possible action of Topo III during transcription will be investigated by exploring fully a recent observation from this laboratory that this enzyme can form a covalent complex with RNA. The ability of Topo III to act catalytically on RNA (i. e., break and reseal strands) will be determined and assays developed to demonstrate Topo III-catalyzed topological modulation of RNA. Topo III can also act to suppress recombination in yeast and E. coli (preliminary data from this laboratory as well as others). It seems likely that the enzyme acts to destabilize some recombinational intermediate. The biochemical basis of this recombination suppression will be approached by examining the effect of Topo III on various joined DNA molecules and RecA-catalyzed strand transfer reactions, as well as on deletion formation and homologous pairing in reconstituted systems in vitro simultaneously undergoing replication (to provide the single strands required for the initiation of recombination) and recombination. It is hoped that at the end of this grant period a more complete picture will have emerged concerning topoisomerase managed trafficking of DNA and RNA strands and in particular of the properties of Topo III that allow it to participate in three major aspects of DNA metabolism: replication, transcription, and recombination.