The overall goal of these studies is to identify and characterize cytotoxic drugs that act by interfering with the regulation of mitochondrial DNA (mtDNA) structure and function. In this regard, we have recently found that nalidixic acid and ciprofloxacin, two 4- quinolone drugs that are known to target at the bacterial type II topoisomerase DNA gyrase, cause a selective loss of mtDNA from mammalian cells. The loss of mtDNA is associated with a decrease in cellular respiration and a loss in cell viability. Analysis of the DNA from 4- quinolone-treated cells revealed the presence of site-specific double- strand breaks in the mtDNA suggesting that mammalian mitochondria may contain a DNA topoisomerase II enzyme that is sensitive to inhibitors of the bacterial type II topoisomerase DNA gyrase. We propose to investigate whether the cytotoxic effects of the 4-quinolone drugs on mammalian cells are mediated through inhibition of a mitochondrial topoisomerase II enzyme, resulting in the loss of mtDNA function. Specifically we propose to: 1. Characterize the effects of topoisomerase II active drugs on mtDNA in cells and in isolated mitochondria. The properties of the drug- induced breaks in mtDNA will be characterized to determine if they are mediated by a mitochondrial topoisomerase. In addition, we will investigate the effects of these drugs on the structure, topology and turnover of mtDNA. 2. Purify and characterize the drug-induced mtDNA cleavage activity from yeast and mammalian cells. 3. Isolate and characterize the cDNA sequences encoding the """"""""putative"""""""" mitochondrial DNA topoisomerase II enzyme from a human cDNA library using universal topoisomerase II probes that contain sequences conserved between eukaryotic or prokaryotic topoisomerase II enzymes. 4. Determine if mtDNA is the primary cytotoxic target of the 4-quinolone drugs (i.e., nalidixic acid and ciprofloxacin) using mutants lacking mitochondrial DNA (mtDNA -) or mutants that are selected for resistance to ciprofloxacin. The mtDNA-mutant(s) will also be used to identify other cytotoxic drugs that act by interfering with the function or replication of mtDNA. These studies should provide important information about the mechanism underlying the cytotoxic effects of 4-quinolones as well as contribute to our basic knowledge regarding the role of topoisomerases in the regulation of mtDNA structure and function.
