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A RADAR method to measure DNA topoisomerase covalent complexes
Alice Meroni1, Alessandro Vindigni1
1Division of Oncology, Department of Medicine, Washington University in St. Louis, St. Louis, MO, United States.
Abstract:
DNA topoisomerases resolve topological stress by introducing transient single- or double-strand breaks into the DNA duplex. This reaction requires the covalent binding of topoisomerases to DNA while the topological stress is being released. This transient intermediate is known as topoisomerase-covalent complex and represents the target of many anti-cancer drugs. Here, we describe a protocol to quantitatively detect topoisomerase-covalent complexes in vivo, called RADAR (rapid approach to DNA adduct recovery). DNA and protein-DNA covalent complexes are rapidly isolated from cells through chaotropic extraction. After normalization, samples are loaded on a slot blot, and the covalent complexes are detected using specific topoisomerase antibodies. In addition to being fast and robust, this assay produces quantitative results. Consequently, the RADAR assay can be applied to investigate the topoisomerase-covalent complex biology, including the effect of specific topoisomerase inhibitors. Finally, the same assay can be more generally applied to study covalent complexes of other enzymes with DNA.
Insights
We developed RADAR, a fast and quantitative method to detect DNA topoisomerase-covalent complexes in vivo. This assay helps study drug effects and DNA adducts from other enzymes.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- DNA topoisomerases manage DNA topology by forming transient covalent complexes.
- These topoisomerase-covalent complexes are crucial intermediates and targets for anti-cancer drugs.
- Existing methods for detecting these complexes are often time-consuming or lack quantitative accuracy.
Purpose of the Study:
- To introduce a novel, rapid, and quantitative protocol for detecting in vivo topoisomerase-covalent complexes.
- To provide a robust assay for studying the biological roles of topoisomerase-DNA interactions.
- To enable the investigation of topoisomerase inhibitor efficacy and mechanisms.
Main Methods:
- Developed the Rapid Approach to DNA Adduct Recovery (RADAR) assay.
- Utilized chaotropic extraction for rapid isolation of DNA and protein-DNA covalent complexes from cells.
- Employed slot blot hybridization with specific topoisomerase antibodies for quantitative detection.
Main Results:
- Demonstrated that RADAR is a fast, robust, and quantitative method for detecting topoisomerase-covalent complexes in vivo.
- Established the utility of RADAR for investigating topoisomerase biology and the effects of inhibitors.
- Showcased the broader applicability of the RADAR assay for studying other enzyme-DNA covalent complexes.
Conclusions:
- The RADAR assay offers a significant advancement in the study of topoisomerase-covalent complexes.
- This method facilitates research into DNA topology, enzyme-DNA interactions, and the development of targeted therapies.
- RADAR provides a versatile platform for investigating various DNA adducts and their biological implications.
