A joint project of the Graduate School, Peabody College, and the Jean & Alexander Heard Library

Title page for ETD etd-03122015-102100

Type of Document Dissertation
Author Lindsey, Jr., Robert Hunter
URN etd-03122015-102100
Title Insights into the Catalytic Mechanism of Eukaryotic and Bacterial Type II Topoisomerases and the Actions of Topoisomerase II Poisons
Degree PhD
Department Biochemistry
Advisory Committee
Advisor Name Title
Neil Osheroff Committee Chair
Daniel Liebler Committee Member
Eric Skaar Committee Member
Martin Egli Committee Member
Nicholas Reiter Committee Member
  • gyrase
  • DNA topology
  • topoisomerase II poisons
  • quinolones
  • topoisomerase II
Date of Defense 2015-03-11
Availability unrestricted
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.

  Filename       Size       Approximate Download Time (Hours:Minutes:Seconds) 
 28.8 Modem   56K Modem   ISDN (64 Kb)   ISDN (128 Kb)   Higher-speed Access 
  LindseyDissertationFinalB.pdf 7.35 Mb 00:34:02 00:17:30 00:15:19 00:07:39 00:00:39

Browse All Available ETDs by ( Author | Department )

If you have more questions or technical problems, please Contact LITS.