The long-term objective of the work in this laboratory is to understand the detailed mechanisms of enzymes that use the chemical energy of nucleotide triphosphates to effect structural changes or movements of enzyme-bound DNA. This proposal addresses the mechanism of the type II DNA topoisomerase. More specifically, these studies are designed to understand how this enzyme utilizes ATP binding/hydrolysis to promote the transport of one DNA duplex through a transient, enzyme-mediated break in another. Apart from being mechanistically challenging and fascinating enzymes, type II topoisomerases are biologically and clinically important. These enzymes are essential in all known organisms and are thought to be required for chromatin condensation and the separation of intertwined chromosomes during both mitosis and meiosis. Through regulating the supercoiling of chromosomal DNA, their activity also affects cell growth and gene expression. They are the targets of a long and growing list of antitumor, antibiotic and anti-fungal drugs, and yet their mechanism of action is largely unknown. The proposed approach is to use a combination of rapid kinetic techniques, mutagenesis and protein chemistry. The single-turnover rates of ATP binding and hydrolysis, as well as DNA transport will be measured under a number of reaction conditions by rapid quench-flow. The mechanism by which phosphorylation of the enzyme increases the steady-state reaction rates will be determined. The comformational changes in the protein and protein-DNA complex that accompany ATP binding, ATP hydrolysis and product dissociation will be followed by stopped-flow fluorescence. Two different types of protein cross-linking experiments will be used to determine whether DNA is transported all the way through the enzyme. Studies on mutant/wild type topoisomerase heterodimers will indicate how the protein conformational changes drive DNA transport. Results from these studies will increase our knowledge of how proteins can couple chemical energy to mechanical work, how topoisomerases may be regulated in the cell, and how to better design topoisomerase II-targeting drugs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051194-04
Application #
2459536
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1994-08-01
Project End
1999-07-31
Budget Start
1997-08-01
Budget End
1998-07-31
Support Year
4
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Utah
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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Stray, James E; Lindsley, Janet E (2003) Biochemical analysis of the yeast condensin Smc2/4 complex: an ATPase that promotes knotting of circular DNA. J Biol Chem 278:26238-48
Lindsley, J E (2001) Use of a real-time, coupled assay to measure the ATPase activity of DNA topoisomerase II. Methods Mol Biol 95:57-64
Baird, C L; Gordon, M S; Andrenyak, D M et al. (2001) The ATPase reaction cycle of yeast DNA topoisomerase II. Slow rates of ATP resynthesis and P(i) release. J Biol Chem 276:27893-8
Morris, S K; Baird, C L; Lindsley, J E (2000) Steady-state and rapid kinetic analysis of topoisomerase II trapped as the closed-clamp intermediate by ICRF-193. J Biol Chem 275:2613-8
Morris, S K; Lindsley, J E (1999) Yeast topoisomerase II is inhibited by etoposide after hydrolyzing the first ATP and before releasing the second ADP. J Biol Chem 274:30690-6
Lindsley, J E (1999) Overexpression and purification of Saccharomyces cerevisiae DNA topoisomerase II from yeast. Methods Mol Biol 94:187-97
Baird, C L; Harkins, T T; Morris, S K et al. (1999) Topoisomerase II drives DNA transport by hydrolyzing one ATP. Proc Natl Acad Sci U S A 96:13685-90
Morris, S K; Harkins, T T; Tennyson, R B et al. (1999) Kinetic and thermodynamic analysis of mutant type II DNA topoisomerases that cannot covalently cleave DNA. J Biol Chem 274:3446-52
Harkins, T T; Lindsley, J E (1998) Pre-steady-state analysis of ATP hydrolysis by Saccharomyces cerevisiae DNA topoisomerase II. 1. A DNA-dependent burst in ATP hydrolysis. Biochemistry 37:7292-8

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