Probing the topoisomerase II mechanism using mutants and inhibitors

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Title Probing the topoisomerase II mechanism using mutants and inhibitors
Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Morris, Shayne Kenneth
Date 2000-12
Description Type II DNA topoisomerases function as molecular motors, utilizing the energy in ATP hydrolysis to pass one DNA duplex through an enzyme-mediated double-stranded break in a second DNA. The enzyme uses a complex mechanism, somehow coupling DNA cleavage/relegation, and transport of a second DNA segment to sequential ATP hydrolysis. In order to define further the entire topoisomerase II mechanism, site directed mutagenesis and topoisomerase II inhibitors were used as mechanistic probes. Additionally, these studies analyzed how the drugs, etoposide and ICRF-193, interrupt the topoisomerase II reaction cycle. The coupling between ATP hydrolysis and DNA cleavage/relegation reactions was studied using mutant enzymes that cannot cleave DNA and etoposide, an anticancer drug that blocks DNA relegation. Blocking DNA cleavage prevents enzyme binding to the transported DNA segment, and reduces the rate of hydrolysis of the first ATP. Blocking DNA relegation inhibits the multiple turnover ATPase and DNA transport reactions by hindering either the first ADP release or the second ATP hydrolysis. These results suggest that DNA cleavage allows the transported DNA segment to bind, stimulating ATP hydrolysis. Additionally, DNA relegation occurs before the second ADP is released, keeping the enzyme in the closed form until the DNA break is resealed. ICRF-193 is an experimental drug that locks topoisomerase II in a closed, intermediate conformation. Surprisingly, this intermediate form continues to hydrolyze ATP, albeit at a reduced rate. Results of rapid kinetic experiments indicate that this drug binds to the enzyme after both ATP have been hydrolyzed, but before the second ADP is released. Together with the cleavage mutant and etoposide data, these results have led to a more complete understanding of the topoisomerase II mechanism.
Type Text
Publisher University of Utah
Subject ATP hydroysis; DNA transport; Topoisomerase; Biochemistry; Molecular Biology
Subject MESH DNA Topoisomerases, Type II; Biochemistry
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "Probing the topoisomerase II mechanism using mutants and inhibitors." Spencer S. Eccles Health Sciences Library. Print version of "Probing the topoisomerase II mechanism using mutants and inhibitors." available at J. Willard Marriott Library Special Collection. QP6.5 2000 .M67.
Rights Management © Shayne Kenneth Morris.
Format application/pdf
Format Medium application/pdf
Format Extent 2,310,615 bytes
Identifier undthes,5232
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Master File Extent 2,310,657 bytes
ARK ark:/87278/s6251m14
Setname ir_etd
ID 191187
Reference URL https://collections.lib.utah.edu/ark:/87278/s6251m14
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