[14] DNA topoisomerases VI from hyperthermophilic archaea

DNA topoisomerases are enzymes that can transiently break the DNA backbone and force the crossing of one or two DNA strands through the other, thereby interconverting DNA topological isomers. They have been classified into two types according to their mechanistic properties. The type I DNA topoisomerases transiently break a single strand of the DNA helix, pass the other strand through this break, and religate the broken strand, thereby altering the linking number by an increment of one. Type II topoisomerases function by cleaving both strands of a DNA molecule, passing an intact double strand through this break, and religating the broken strands thereby changing the linking number by an increment of two. The DNA topoisomerases VI are type II DNA topoisomerases only found in archaea. These enzymes exhibit all the biochemical properties of type II DNA topoisomerases. This chapter presents the purification of the DNA topoisomerase VI from Pyrococcus furiosus, an extremely thermophilic anaerobic euryarchaeota with an optimal growth temperature of 95°. These two enzymes share many properties and can be purified by a similar procedure. However, the purification of the DNA topoisomerase VI from P. furiosus has been simplified and some modifications, required by the extreme thermophily of this enzyme, have been introduced into DNA topoisomerase assays. Finally, physical and enzymatic properties of DNA topoisomerase VI are discussed.