18 Type I DNA Topoisomerases

Abstract

DNA topoisomerases are enzymes that catalyze the breakage and rejoining of DNA strands. The breakage of a DNA backbone bond by a topoisomerase is followed efficiently by the rejoining of the same bond. Therefore, the disjoined state is a transient one and each cycle of breakage and rejoining of a bond is almost invariably detected by the conversion of one DNA topological isomer to another. DNA topoisomerases can be divided into two categories—namely, type I and type II. This chapter focuses on type I DNA topoisomerases. The type I enzymes catalyze the breakage and rejoining of DNA strands one at a time. They require no energy cofactors such as ATP or NAD. The purification of the bacterial DNA topoisomerase enzyme follows the steps of cell lysis, nucleic acid removal, ammonium sulfate precipitation, and chromatographic separations. The relaxation of a negatively supercoiled DNA is commonly used to monitor the enzymatic activity during purification. The purification of the eukaryotic type I topoisomerases involves the isolation of cell nuclei as the initial step. Subsequent fractionations by polyethylene glycol extraction and chromatography are commonly carried out. The earlier preparations yield typically proteins with molecular weights of about 65,000.