DNA Decatenation Catalyzed by Bacterial Topoisomerase IV

Abstract

One of the essential functions of type II topoisomerases is the decatenation (unlinking) of interlinked daughter chromosomes that are formed due to the torsional stress generated by the opening of the double helix during DNA replication. To resolve these intermolecular tangles, type II topoisomerases interact with two separate DNA molecules and pass one through the other. Different topoisomerases can catalyze this reaction with variable efficiency, and decatenation activity can be measured in vitro using purified enzymes and a catenated DNA substrate. The essential decatenation function of type II topoisomerases can also be inhibited by antibacterial and anticancer compounds. This chapter will describe an agarose gel electrophoresis-based assay to measure the decatenation of kinetoplast DNA purified from Crithidia fasciculata by bacterial topoisomerase IV. It will also describe how the assay can be used to monitor the inhibition of decatenation by topoisomerase IV-targeted antibacterial drugs. Finally, the protocols described in this chapter can be easily modified for use with other bacterial and eukaryotic type II topoisomerases.

Keywords: Antibacterials; DNA catenation; DNA decatenation; DNA topoisomerase IV; Escherichia coli; Kinetoplast DNA; Replication; kDNA.

Figure 1.. Representative gel image of a decatenation assay time course

Kinetoplast DNA (kDNA) is such a large catenated complex that it is unable to migrate out of the wells (top) of a 1% agarose gel (DNA Control). Over the course of the assay, topoisomerase IV decatenates the maxi- and mini-circles that make up the kDNA, which allows them to migrate into the gel (bottom). The decatenated nicked and supercoiled mini-circles are especially visible. This results in the accumulation of decatenated products and a decrease in fluorescence intensity in the wells.

Figure 2.. Representative gel image displaying the effects of an antibacterial on decatenation.

Kinetoplast DNA (kDNA) is such a large catenated complex that it is unable to migrate out of the wells (top) of a 1% agarose gel (DNA Control). In the absence of an inhibitor, topoisomerase IV can decatenate the maxi- and mini-circles that compose kDNA allowing them to migrate into the gel (Enzyme Control). The decatenated nicked and supercoiled mini-circles are especially visible. As the concentration of an antibacterial ([Compound]) increases, the ability of the enzyme to catalyze decatenation decreases. This results in a loss of decatenated products accompanied by an increased quantity of kDNA in the wells.