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.
Highlights
DNA topoisomerases are essential, ubiquitous enzymes that maintain the topological integrity of the genome [1,2,3,4,5,6,7,8,9,10,11]
Type II topoisomerases cleave both strands of the helix to relieve torsional stress and maintain the appropriate level of supercoiling, and to resolve knots and tangles that arise as a result of normal cellular processes and decatenate daughter chromosomes during replication [1,5,6,12,13,14,15,16,17,18,19]
“lysis at 37 ◦ C was crucial for good yield of covalent complex and that lysis at 4 ◦ C or 0 ◦ C yielded negative results” [35]
Summary
DNA topoisomerases are essential, ubiquitous enzymes that maintain the topological integrity of the genome [1,2,3,4,5,6,7,8,9,10,11]. Type II topoisomerases cleave both strands of the helix to relieve torsional stress and maintain the appropriate level of supercoiling, and to resolve knots and tangles that arise as a result of normal cellular processes and decatenate daughter chromosomes during replication [1,5,6,12,13,14,15,16,17,18,19] Due to their mechanisms of action that require breaking the DNA backbone, these enzymes are inherently dangerous to the cell
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