Regulation of the catalytic cycle of topoisomerase II
Topoisomerase II is an essential enzyme that modulates DNA topology in vivo. The catalytic cycle of this enzyme can be dissected into at least six discrete steps: (1) enzyme[center dot]DNA binding; (2) pre-strand passage DNA cleavage/religation; (3) DNA strand passage; (4) post-strand passage DNA cleavage/religation; (5) ATP hydrolysis; and (6) enzyme turnover. Each activity catalyzed by topoisomerase II has the potential to be a control point for the regulation of overall enzyme activity. The present work demonstrates that the initial topoisomerase II-mediated DNA cleavage event is regulated by the enzyme's interaction with its DNA substrate. The enzyme requires that both the cleavage helix and the passage helix be present in the enzyme[center dot]DNA complex prior to DNA cleavage. In addition, results demonstrate that the catalytic activity of topoisomerase II is altered by the presence of cyclobutane pyrimidine dimers in substrate DNA. A novel non-turnover DNA catenation assay was developed to demonstrate that these DNA lesions inhibit overall catalytic activity by decreasing the ability of the enzyme to carry out its DNA strand passage event. Finally, phosphorylation stimulates the catalytic activity of topoisomerase II specifically by enhancing the ATP hydrolysis step of the catalytic cycle. Phosphorylation mediated by either casein kinase II or protein kinase C enhances overall enzyme activity by the same mechanism. All of these studies examine the individual steps of the catalytic cycle of topoisomerase II to determine the exact mechanism by which effectors alter enzyme activity. These represent the first studies that employ this approach to determine specific control points within the catalytic cycle of the enzyme. With the development of an assay for the DNA strand passage event of topoisomerase II, it is now possible to examine each step of the catalytic cycle that precedes enzyme turnover.
- Research Organization:
- Vanderbilt Univ., Nashville, TN (United States)
- OSTI ID:
- 7296661
- Resource Relation:
- Other Information: Thesis (Ph.D.)
- Country of Publication:
- United States
- Language:
- English
Similar Records
Investigating mycobacterial topoisomerase I mechanism from the analysis of metal and DNA substrate interactions at the active site
Intermolecular DNA ligation activity of eukaryotic toposiomerase II: Potential roles in nucleic acid recombination
Related Subjects
37 INORGANIC
ORGANIC
PHYSICAL AND ANALYTICAL CHEMISTRY
DNA
ISOMERIZATION
ISOMERASES
CATALYTIC EFFECTS
ENZYME ACTIVITY
BIOCHEMICAL REACTION KINETICS
CYCLOALKANES
PHOSPHORYLATION
ALKANES
CHEMICAL REACTIONS
ENZYMES
HYDROCARBONS
KINETICS
NUCLEIC ACIDS
ORGANIC COMPOUNDS
PROTEINS
REACTION KINETICS
550200* - Biochemistry
400201 - Chemical & Physicochemical Properties