The long-term objectives of my research are to understand the detailed mechanisms of mitochondrial biogenesis at the level of enzyme structure and function as well as at the level of mtDNA organization and expression. The specific goal of this proposal is to begin to elucidate the roles of DNA topoisomerases in the replication and metabolism of mtDNA. In particular, the interaction of ATP with nuclear and mitochondrial type I topoisomerase will be investigated. It has been shown that the ATP-independent type I topoisomerase is actually inhibited by ATP. Since this effect has been observed with topos from human leukemia, HeLA and calf thymus cells, we hypothesize that this ATP regulation of topo I activity may be a general phenomenon in mammalian cells. The details of the nucleotide-enzyme interaction as well as the mechanism of this regulation using in vivo and in vitro studies will be elucidated. The role of these enzymes in mitochondrial DNA replication and metabolism will be defined by investigating the interactions of topoisomerase I and II with the mitochondrial genome in vivo and in vitro. These studies will utilize several antitumor drugs which have been shown to specifically interfere with the topoisomerase- catalyzed strand breaking and rejoining process such that, in the presence of a protein denaturant, DNA cleavage results with topoisomerase covalently attached to the end of the DNA fragment. In particular, camptothecin will be used to probe for topo I-DNA interactions, and 4'- (9-acridinylamin)-methane sulfon-m-anisidide (mAMSA) and the epipodophyllotoxins VP-16 and VM-26 will be used to prove topo II-DNA interactions. These studies should elucidate some of the details of the involvement of topo I and topo II in replication and expression of the mitochondrial genome. Regulation of topoisomerase activity is of general significance but as detailed in the proposal, the maintenance of proper chromosomal and extrachromosomal DNA superhelicity is important in carcinogenesis, mutagenesis and tumorigenesis and as such these studies are relevant to problems in cancer etiology and therapy.
