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.