A RADAR-Based Assay to Isolate Covalent DNA Complexes in Bacteria

Abstract

Quinolone antibacterials target the type II topoisomerases gyrase and topoisomerase IV and kill bacterial cells by converting these essential enzymes into cellular poisons. Although much is known regarding the interactions between these drugs and enzymes in purified systems, much less is known regarding their interactions in the cellular context due to the lack of a widely accessible assay that does not require expensive, specialized equipment. Thus, we developed an assay, based on the "rapid approach to DNA adduct recovery," or RADAR, assay that is used with cultured human cells, to measure cleavage complex levels induced by treating bacterial cultures with the quinolone ciprofloxacin. Many chemical and mechanical lysis conditions and DNA precipitation conditions were tested, and the method involving sonication in denaturing conditions followed by precipitation of DNA via addition of a half volume of ethanol provided the most consistent results. This assay can be used to complement results obtained with purified enzymes to expand our understanding of quinolone mechanism of action and to test the activity of newly developed topoisomerase-targeted compounds. In addition, the bacterial RADAR assay can be used in other contexts, as any proteins covalently complexed to DNA should be trapped on and isolated with the DNA, allowing them to then be quantified.

Keywords: ICE assay; Quinolone; RADAR assay; covalent complex; gyrase; topoisomerase.

Figure 1

Catalytic cycle of type II topoisomerases. Step 1: The enzyme bends the gate-, or G-, segment of DNA in the presence of divalent metal ions (the physiological ion is Mg²⁺). Step 2: The enzyme cleaves and covalently attaches to the newly generated 5’-termini of the G-segment, generating the cleavage complex. Step 3: The enzyme passes the transfer-, or T-, segment of DNA through the cut it generated in the G-segment. Step 4: The enzyme religates the G-segment. Step 5: The T-segment is released from the enzyme. Step 6: The enzyme releases the G-segment and resets for another round of catalysis. Note that ATP hydrolysis is required for the enzyme to complete the catalytic cycle. Modified from Reference [22].

Figure 2

Flow chart outlining methods 19 (on the left branch) and 16 (on the right branch). Equivalent steps are aligned to provide for easier comparison of the similarities and differences in the two protocols.

Figure 3

Immunoblot and quantification of ciprofloxacin-induced topoisomerase IV cleavage complexes trapped in E. coli. (a) Immunoblot comparing methods 16 and 19. Ciprofloxacin concentrations are listed across the top. Three independent experiments of methods 16 and 19 are shown as indicated at the left. In each case, 100 ng of DNA was blotted. To facilitate quantification, 2, 20, 60, and 100 ng of purified E. coli topoisomerase IV subunit A were also blotted. (b) Quantification of method 19 from (a). (c) Quantification of method 16 from (a). For both (b,c), a standard curve was generated from the topoisomerase IV standards and used to determine the ng of topoisomerase IV present in each band. This amount was then scaled to determine the number of ng of topoisomerase IV per µg of DNA. Error bars represent the standard deviation of three independent experiments.

Figure 4

Representative immunoblots of ciprofloxacin-induced type II topoisomerase cleavage complexes trapped in S. aureus using either method 16 or method 19 as indicated at the left. (a) Immunoblot of ciprofloxacin-induced gyrase cleavage complexes. Purified S. aureus gyrase subunit A (1, 20, and 80 ng) was also blotted. (b) Immunoblot of ciprofloxacin-induced topoisomerase IV cleavage complexes. Purified S. aureus topoisomerase IV subunit A (2.5, 10, and 40 ng) was also blotted. For both (a,b), ciprofloxacin concentrations present in each sample are indicated at the top. A total of 100 ng of DNA was blotted in each case.

Figure 5

Ciprofloxacin-induced intracellular cleavage complex formation by gyrase in E. coli as measured using method 19 (a) and method 16 (b). Quantification was carried out as described in Figure 3 using a simultaneously blotted purified E. coli gyrase subunit A standard. In (a), error bars represent the standard error of the mean of two independent experiments. In (b), error bars represent the standard deviation of three independent experiments.