DNA Supercoiling Catalyzed by Bacterial Gyrase

Abstract

DNA gyrase is an essential type II topoisomerase that is conserved across bacteria species and has an essential function of resolving overwound (positive supercoiled) DNA and introducing negative supercoils into relaxed DNA. The overall catalytic activity of gyrase can be determined using in vitro assays utilizing purified enzyme subunits and a DNA substrate. As gyrase is the only topoisomerase that can introduce negative supercoils into relaxed DNA, the inhibition of this catalytic activity is a good indicator of the efficacy and potency of potential antibacterial compounds. This chapter outlines a protocol for a purified enzyme assay with relaxed DNA that utilizes gel electrophoresis to monitor the ability of gyrase to introduce negative supercoils into DNA. The protocol focuses on the DNA supercoiling activity of Escherichia coli wild-type gyrase. However, it can be easily modified for use with gyrase from other bacterial species.

Keywords: Bacterial type II topoisomerase; DNA Gyrase; DNA supercoiling; Escherichia coli; Negative supercoiling; Overwinding; Positive supercoiling.

Figure 1:

Representative gel showing negatively supercoiled [(−) SC] and relaxed DNA.

Figure 2:

A) Overview of the time course experimental set up. B) Representative gel of a time course showing the DNA supercoiling activity of gyrase. The first lane contains a DNA control with just relaxed DNA. The second lane is a 0 min time point that is taken immediately after incubating the master mixture assay tube at 37°C. Each successive lane has increased levels of negative supercoiled [(−) SC] DNA as the reaction time is increased.

Figure 3:

Representative gel showing a drug titration for the inhibition of the DNA supercoiling activity of gyrase. The first lane shows a DNA control that contains only relaxed DNA, and the second lane contains relaxed DNA and the enzyme without any drug. The next lanes contain an increasing concentration of drug, which inhibits the ability of gyrase to introduce negative supercoils [(−) SC] into the relaxed DNA.