Abstract

Type II DNA topoisomerases are essential enzymes that unlink intertwined chromosomes. By altering DNA topology they participate in chromosome segregation, condensation, replication, recombination and transcription. These enzymes are the targets of numerous clinically-used and trial-phase anticancer and antibiotic drugs, making the understanding of their mechanism medically relevant. The reaction these enzymes catalyze is the ATP-dependent transport of one segment of DNA through a transient break in a second segment of DNA. Recent pre-steady-state ATPase results indicate that the homodimeric topoisomerase II binds two ATP to initiate the reaction cycle, but hydrolyzes only one rapidly. The second ATP is hydrolyzed later in the reaction cycle, after a rate-determining step in the reaction. This strict sequential hydrolysis of the two ATP was completely unexpected and does not agree with any previous models for the mechanism of this enzyme. The goals of the present proposal are to further elucidate the ATPase mechanism, analyze the protein-DNA complex conformational changes during the reaction cycle, and determine when in this cycle DNA transport actually occurs. By combining all of these pre-steady-state and steady- state results, a more accurate mechanism for DNA topoisomerase II catalyzed reactions will be determined.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051194-09
Application #
6525822
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Jones, Warren
Project Start
1994-08-01
Project End
2004-07-31
Budget Start
2002-08-01
Budget End
2004-07-31
Support Year
9
Fiscal Year
2002
Total Cost
$201,743
Indirect Cost

  Institution

Name
University of Utah
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112

  Publications

Stray, James E; Crisona, Nancy J; Belotserkovskii, Boris P et al. (2005) The Saccharomyces cerevisiae Smc2/4 condensin compacts DNA into (+) chiral structures without net supercoiling. J Biol Chem 280:34723-34
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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