|Keywords:||gyrase; DNA topology; topoisomerase II poisons; quinolones; topoisomerase II|
|Full text PDF:||http://etd.library.vanderbilt.edu/available/etd-03122015-102100/|
Coordination between the N-terminal gate and the catalytic core of topoisomerase II allows the proper capture, cleavage, and transport of DNA during catalysis. Because the activities of these domains are tightly linked, it has been difficult to discern their individual contributions to enzyme-DNA interactions and drug mechanism. To address the roles of these domains, the activity of the human topoisomerase IIα catalytic core was analyzed. The catalytic core and full-length enzyme both maintained higher levels of cleavage with negatively vs. positively supercoiled plasmid. Thus, the ability to distinguish supercoil handedness is embedded within the catalytic core. However, the catalytic core displayed little ability to cleave DNA substrates that did not intrinsically provide the enzyme with a transport segment (substrates that did not contain crossovers). Therefore, the N-terminal gate is critical for the capture of the T-segment. In contrast to interfacial topoisomerase II poisons, covalent poisons did not enhance DNA cleavage mediated by the catalytic core. This distinction allowed further characterization of etoposide quinone, a drug metabolite that acts primarily as a covalent poison. Etoposide quinone retained some ability to enhance DNA cleavage mediated by the catalytic core, indicating that it still can function as an interfacial poison. Gyrase removes positive supercoils ahead of replication forks in bacteria. However, nothing is known about the effects of supercoil geometry on DNA cleavage mediated by gyrase. Similar to the human type II enzymes, Bacillus anthracis gyrase could discern supercoil geometry, and maintained lower levels of cleavage complexes with positively supercoiled substrates. Interactions of quinolones with topoisomerase IV are mediated through a water-metal ion bridge that is anchored in part by a conserved serine residue (Ser81 B. anthracis gyrase). However, the role of the bridge appears to vary between species and has not been assessed in gyrase. Therefore, the sensitivity of wild-type and GyrAS85L gyrase towards quinolones and quinazolinediones was assessed. Preliminary findings support the hypothesis that gyrase uses the water-metal ion bridge to coordinate quinolone interactions and pave the way for future mechanistic studies.