Topoisomerase are ubiquitous enzymes that alter the linking number of DNA. As such, they play essential roles in every aspect of DNA metabolism. Their importance is underscored by the fact that in eukaryotes these enzymes are the cellular targets of potent anticancer drugs, whereas in prokaryotes both DNA Gyrase and topoisomerase IV (Topo IV) are targets of the most potent broad-spectrum antibacterial agents (e.q., ciprofloxacin). These drugs convert topoisomerases to poisons that inhibit DNA replication and lead to double-strand break (DSB) generation. Thus, it is crucial to understand the molecular basis of the cytotoxicity of these topoisomerase inhibitors. I have shown that the encounter of a replication fork with a Topo IV- quinolone-DNA ternary complex converts the ternary complex to a nonreversible form. (I) We will examine if a complete replication fork is required for the conversion of a ternary complex to a nonreversible form. It is possible that the DnaB helicase alone is the active agent. (II) We will study the effects of a topoisomerase trapped at a site of DNA damage on the replication fork. It has been demonstrated that DNA lesions, such as an apurinic site, stimulate eukaryotic topoisomerase- mediated DNA cleavages. We will examine if an apurinic site also acts as a topoisomerase poison for bacterial enzymes. We will compare effects of an apurinic site-induced Topo IV-DNA complex and a Topo IV- quinolone-DNA ternary complex on the replication fork to determine if these two distinct mechanisms of poisoning topoisomerases have the same consequences. (III) Recent studies have demonstrated that quinolones do not stimulate S. aureus DNA gyrase (Gyrase)-mediated DNA cleavages. We will characterize S. aureus Gyrase and a ternary complex formed with this enzyme. Furthermore, we will examine if DNA strand cleavage is always required for replication fork arrest by a topoisomerase- quinolone-DNA ternary complex. (IV) We have proposed that quinolone- induced DSB generation is a two-step process. Using an assay where replication forks collide with a ternary complex, I detected an activity in an E. coli extract that could generate DSBs at dead-end topoisomerase complexes. We will identify the protein(s) required for the second step of DSB formation and complete the reconstitution of the DSB process in vitro with purified-proteins.
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