Topoisomerases are required for every aspect of DNA replication. The type II enzymes are the target of very potent chemothearaputic agents in both prokaryotes and eukaryotes-the quinolone family of antibacterials, including ciprofloxacin, one of the most widely prescribed antimocrobials in this country, and the epipodophyllotoxins, a family of very potent anticancer agents that includes the drug Etoposide. Thus, understanding how Topoisomerases work and how their activities relate to and are required by other cellular processes is crucial both for our understanding of DNA metabolism in the cell and in the development of more effective clinical interventions. Genes encoding four of the ten subunits of the holoenzyme have been found to suppress both the temperature-sensitive and partition phenotypes of either mutant parC or parE alleles. These genes encode the subunits of Topo IV. The investigator and his group propose that this indicates that Topo IV is present in the cell in a complex with the holoenzyme, probably at the replication fork. This will be investigated by a combined biochemical and cell biological approach to detect physical and functional interactions between these proteins and to localize them in the cell. Dr. Marians and his group have also isolated a gene encoding a putative inner membrane protein of unknown function as a high-copy suppressor of a mutant parC allele. This gene does not suppress mutation in gyrB. They propose that the suppressor protein serves as a membrane anchor for Topo IV in the cell. Biochemical techniques will be used to study the distribution of the suppressor protein in the cell and whether it interacts with Topo IV in vitro. Genetic techniques will be used to inactivate the gene encoding the parC suppressor protein and the consequent phenotypes examined. In situ hybridization and immunofluorescence microscopy will be used to determine whether the suppressor protein and Topo IV colocalize in the cell. Quinolone cytotoxicity is associated with the appearance of DNA double-strand breaks, yet the mechanism of their formation is unknown. Dr. Marians and his group have shown that two steps are required: collision of a replication fork with a quinolone-Topo IV-DNA complex to yield a nonreversible complex and then the subsequent action of other cellular proteins to generate the double-strand breaks. Biochemical techniques will be used to isolate the proteins responsible for double-strand break-formation at these nonreversible complexes.
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