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Atp-bound topoisomerase ii as a target for antitumor drugs
1Department of Pharmacology, University of Medicine and Dentistry of New Jersey/Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Abstract:
Topoisomerase II (TOP2) poisons interfere with the breakage/reunion reaction of TOP2 resulting in DNA cleavage. In the current studies, we show that two different classes (ATP-sensitive and -insensitive) of TOP2 poisons can be identified based on their differential sensitivity to the ATP-bound conformation of TOP2. First, in the presence of 1 mm ATP or the nonhydrolyzable analog adenosine 5'-(beta,gamma-imino)triphosphate, TOP2-mediated DNA cleavage induced by ATP-sensitive TOP2 poisons (e.g. doxorubicin, etoposide, mitoxantrone, and 4'-(9-acridinylamino)methanesulfon-m-anisidide) was 30-100-fold stimulated, whereas DNA cleavage induced by ATP-insensitive TOP2 poisons (e.g. amonafide, batracylin, and menadione) was only slightly (less than 3-fold) affected. In addition, ADP was shown to strongly antagonize TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive TOP2 poisons. Second, C427A mutant human TOP2alpha, which exhibits reduced ATPase activity, was shown to exhibit cross-resistance to all ATP-sensitive but not ATP-insensitive TOP2 poisons. Third, using ciprofloxacin competition assay, TOP2-mediated DNA cleavage induced by ATP-sensitive but not ATP-insensitive poisons was shown to be antagonized by ciprofloxacin. These results suggest that ATP-bound TOP2 may be the specific target of ATP-sensitive TOP2 poisons. Using Lac repressor-operator complexes as roadblocks, we show that ATP-bound TOP2 acts as a circular clamp capable of entering DNA ends and sliding on unobstructed duplex DNA.
Insights
Two classes of topoisomerase II (TOP2) poisons exist: ATP-sensitive and ATP-insensitive. ATP-sensitive poisons specifically target the ATP-bound conformation of TOP2, influencing DNA cleavage and drug resistance.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Topoisomerase II (TOP2) is crucial for DNA replication and repair.
- TOP2 poisons induce DNA cleavage by interfering with TOP2's breakage/reunion activity.
- Understanding TOP2 poison mechanisms is vital for cancer therapy.
Purpose of the Study:
- To differentiate TOP2 poisons based on their interaction with TOP2's ATP-bound state.
- To identify the specific target of ATP-sensitive TOP2 poisons.
- To elucidate the structural dynamics of ATP-bound TOP2.
Main Methods:
- Assessing TOP2-mediated DNA cleavage in the presence of ATP analogs (ATP, АТРγ-imido), ADP, and specific TOP2 poisons.
- Utilizing a C427A mutant human TOP2alpha with reduced ATPase activity.
- Employing ciprofloxacin competition assays.
- Analyzing TOP2-DNA interactions using Lac repressor-operator complexes.
Main Results:
- ATP-sensitive poisons (doxorubicin, etoposide) showed 30-100-fold stimulation of DNA cleavage with ATP, while ATP-insensitive poisons (amonafide, batracylin) showed minimal effect (<3-fold).
- ADP antagonized cleavage by ATP-sensitive poisons but not ATP-insensitive ones.
- The C427A TOP2alpha mutant exhibited cross-resistance to ATP-sensitive poisons.
- Ciprofloxacin antagonized cleavage by ATP-sensitive but not ATP-insensitive poisons.
- ATP-bound TOP2 functions as a circular clamp on DNA.
Conclusions:
- ATP-bound TOP2 is the specific target of ATP-sensitive TOP2 poisons.
- This classification provides a new framework for understanding TOP2 poison mechanisms.
- The findings have implications for developing novel anticancer drugs targeting TOP2.