Rapid, direct detection of bacterial topoisomerase 1-DNA adducts by RADAR/ELISA

• Published in: Analytical biochemistry Vol. 608; p. 113827
• Main Authors: Sinha, Devapriya, Kiianitsa, Kostantin, Sherman, David R., Maizels, Nancy
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2020
• Subjects: Antibiotic; Bacterial Proteins - analysis; Bacterial Proteins - biosynthesis; Bacterial Proteins - isolation & purification; Cell Fractionation - methods; DNA Adducts - analysis; DNA Adducts - isolation & purification; DNA Topoisomerases, Type I - analysis; DNA Topoisomerases, Type I - isolation & purification; DNA-Protein crosslink; Enzyme-Linked Immunosorbent Assay - methods; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Gyrase; High-Throughput Screening Assays - methods; Immunoblotting - methods; Mycobacteria; Mycobacterium smegmatis - chemistry; Mycobacterium smegmatis - genetics; Mycobacterium smegmatis - metabolism; Reproducibility of Results; Topoisomerase poison; Tuberculosis; Yersinia pestis - genetics; RADAR; Mycobacteria; Ms; Mt; ATc; Antibiotic; Tuberculosis; Topoisomerase poison; CFU; Gyrase; GTC; Yp; LSB; DNA-Protein crosslink; Ec; ELISA
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2020.113827

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. pestis and M. tuberculosis, expressed in E. coli or M. 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. [Display omitted] •RADAR/ELISA enables simple and convenient quantification of Top1-DNA adducts in living bacteria.•Mechanism-based assay makes bacterial Top1 an accessible target for drug discovery.•RADAR fractionation disrupts and kills even normally recalcitrant Mycobacteria.