In vitro assays used to measure the activity of topoisomerases

Abstract

INTRODUCTIONAn earlier review discussed the multitude of biochemical reactions carried out by topoisomerases in the maintenance of DNA topology in cells (86). An understanding of these reactions has been obtained through the use of various assays that utilize gel electrophoresis techniques to separate the DNA products resulting from topoisomerase action on a DNA substrate. Staining of these DNA species with ethidium bromide has enabled the development of methods to measure the fluorescence of the ethidium bromide-DNA complex, making the topoisomerase assays more quantitative, the testing of chemical inhibitors more standardized, and the results more comparable between laboratories. While numerous assays to measure the activity of topoisomerases have been described in the literature (14,20,42,44,49,65,86,95,99,100), all of them can be divided into one of two groups, depending on the activity measured. The first group of assays measures the catalytic activity of topoisomerases and determines the extent of inhibition of relaxation (7, 97), supercoiling (2, 22, 23, 65, 83, 85), unknottingknotting (43, 44), or decatenation-catenation (33,39,40,73). The noncatalytic assays are designed to measure the proteinassociated DNA breaks (cleavage) that represent the reversible breakage and reunion activity of topoisomerases (10,17,28,36,45,56,74,81,96,101). The purpose of this review is to discuss the design and experimental conditions of the most frequently used topoisomerase assays and to consider their application in measuring the effects of quinolone antibacterial agents on both procaryotic and eucaryotic topoisomerases.CATALYTIC ASSAYS Topoisomerase I can be purified from both procaryotic and eucaryotic sources; calf thymus topoisomerase I can be purchased commercially (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). Commercial eucaryotic topoisomerase I is frequently used to prepare the relaxed DNA substrate for the supercoiling reaction (83). The two subunits of procaryotic topoisomerase II (gyrase) can be purified from a pair of Escherichia coli strains constructed by Mizuuchi et al. (54) to overexpress the gyrase A and B subunits. Alternatively, Micrococcus luteus gyrase can be purchased commercially (Bethesda Research Laboratories). Purification of the subunits separately from such recombinant strains is usually preferred over purification of the holoenzyme (19,54,65,82,85 (20,55,62,86,95,98,99) (20,23,40,65,67,85), except for the relaxation reaction of DNA gyrase, which is ATP independent. Topoisomerase II exhibits a relatively broad pH optimum from 7.5 to 9.0 in Tris hydrochloride buffers (19,23,40,50,65,85) that contain a reducing agent such as 2 to 5 mM dithiothreitol (23,40,65,85), a condensing agent such as 5 mM spermidine (23,40,65,85) (2,4,22,98). The supercoiled substrate and its relaxed product can be easily distinguished in ethidium bromidestained agarose gels, since the relaxed topological isomers of DNA migrate more slowly than the supercoiled DNA species (Fig. 1, lanes 1 and 2).Relaxation med...

Quinolone-Binding Pocket of DNA Gyrase: Role of GyrB

“…4 Affinity of CFX and OXO for mutant gyrase-DNA complexes. The apparent affinity of quinolone drugs for gyrase-DNA complexes can be measured using the DNA cleavage reaction (4). Quinolones are known to bind to the gyrase-DNA complex and stabilize a form of the complex in which the DNA is cleaved across both strands; in the absence of drug, cleavage is not detectable.…”

Dual Targeting of Topoisomerase IV and Gyrase To Reduce Mutant Selection: Direct Testing of the Paradigm by Using WCK-1734, a New Fluoroquinolone, and Ciprofloxacin

“…Quinolone resistance occurs stepwise by mutations in the two topoisomerase target enzymes, with the first mutation generally occurring in the more sensitive enzyme (13). From the firststep mutants second-step double mutants can then be selected with resistance mutations in the second target enzyme, thereby conferring a high-level resistance phenotype (1,7,21). If the original sensitivities of both DNA gyrase and TopoIV were the same (i.e., dual targeting), no single mutational alteration in either enzyme would result in an increase in the MIC (11,25).…”

Usual and unusual antibacterial effects of quinolones

“…Do quinolones exert their effect by binding to DNA gyrase? Such a hypothesis can be supported by several observations: inhibition of the enzymatic activity of purified DNA gyrase by quinolones 7 Y. X. Fnret and J.-C Pectere (Barrett, Sutcliffe & Gootz, 1990), bacterial resistance associated with DNA gyrase mutations (Gellert et al, 1977;Sato et al, 1986) and "F nuclear resonance experiments showing the affinity of pefloxacin for DNA gyrase (Le Gome, 1985). However, if quinolones bind directly to DNA gyrase, the exact binding site remains to be determined.…”