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A RADAR-Based Assay to Isolate Covalent DNA Complexes in Bacteria.
Katie J Aldred1, Adeline Payne2, Olivia Voegerl3
1Biology Department, University of Evansville, Evansville, IN 47722, USA. ka59@evansville.edu.
Antibiotics (Basel, Switzerland)
|March 2, 2019
Summary
Researchers developed a bacterial RADAR assay to measure quinolone drug effects on bacterial enzymes. This accessible method quantifies drug-target interactions within cells, advancing understanding of antibacterial mechanisms.
Area of Science:
- Microbiology
- Molecular Biology
- Pharmacology
Background:
- Quinolone antibacterials inhibit bacterial type II topoisomerases, gyrase and topoisomerase IV.
- Understanding drug-enzyme interactions in vivo is limited by the lack of accessible assays.
- Existing methods often require expensive, specialized equipment.
Purpose of the Study:
- To develop a novel, accessible assay for measuring quinolone-induced DNA cleavage complexes in bacterial cells.
- To adapt the human cell-based RADAR assay for bacterial applications.
- To facilitate the study of quinolone mechanism of action and the evaluation of new topoisomerase-targeting compounds.
Main Methods:
- Adaptation of the "rapid approach to DNA adduct recovery" (RADAR) assay for bacterial cultures.
- Optimization of lysis and DNA precipitation conditions, including sonication and ethanol precipitation.
- Quantification of drug-induced topoisomerase-DNA cleavage complexes.
Main Results:
- A robust bacterial RADAR assay was established using sonication in denaturing conditions and ethanol precipitation.
- The assay demonstrated consistent results for measuring cleavage complex levels.
- The method is adaptable for studying various protein-DNA interactions.
Conclusions:
- The bacterial RADAR assay provides an accessible tool to study quinolone mechanism of action in a cellular context.
- This assay can complement purified enzyme studies and aid in the development of new antibacterial agents.
- The methodology has broader applications for quantifying any protein covalently bound to DNA within cells.
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