Analysis of eukaryotic DNA topoisomerases and topoisomerase-directed drug effects.

DNA topoisomerases are enzymes which control DNA topology by cleaving and rejoining DNA strands and passing other DNA strands through the transient gaps. Consequently these enzymes play a crucial role in the regulation of the physiological function of the genome. Beyond their normal functions, topoisomerases are important cellular targets in the treatment of human cancers. Some of the most powerful anti-cancer drugs used clinically stabilize the catalytic topoisomerase-DNA intermediates and, thus, cause DNA disorders that will induce apoptosis in proliferating cells. This review summarizes current protocols for measuring the catalytic activity of topoisomerases and for monitoring the molecular effects of topoisomerase-directed antitumour drugs in living cells and in cell-free assays. Furthermore, preanalytical factors are discussed, such as enzyme stability, methods for extracting DNA topoisomerases from cells, and protocols for separating subtypes and isoforms of these enzymes.