Rapid, direct detection of bacterial Topoisomerase 1-DNA adducts by RADAR/ELISA
Autor: | Devapriya Sinha, Nancy Maizels, David R. Sherman, Kostantin Kiianitsa |
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Rok vydání: | 2020 |
Předmět: |
Lysis
Yersinia pestis Immunoblotting Mycobacterium smegmatis Mutant Biophysics Enzyme-Linked Immunosorbent Assay Cell Fractionation 01 natural sciences Biochemistry DNA gyrase Article Adduct DNA Adducts 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins Escherichia coli Molecular Biology 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology 030306 microbiology Topoisomerase 010401 analytical chemistry Reproducibility of Results Cell Biology 0104 chemical sciences High-Throughput Screening Assays 3. Good health Chaotropic agent Enzyme DNA Topoisomerases Type I chemistry biology.protein Nucleic acid DNA |
Zdroj: | Anal Biochem |
DOI: | 10.1101/2020.03.09.984153 |
Popis: | Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by “RADAR fractionation”, a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens,Y. pestisandM. tuberculosis, expressed inE. coliorM. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches. |
Databáze: | OpenAIRE |
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