Purification and characterization of human DNA topoisomerase III[alpha]

Human topoisomerase III[alpha] (hTopo III[alpha]), the recently identified first member of the topoisomerase IA subfamily in humans, has a central domain which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In order to determine the properties of hTopo III[alpha], compared to those of other topoisomerase IA subfamily members, we purified this enzyme to near homogeneity, together with an active site-mutant Y337F. We show that hTopo III[alpha] is able to relax negatively supercoiled DNA in a distributive manner, leading to the total disappearance of the initial substrate and the appearance of intermediate topoisomers. This DNA relaxation activity is magnesium-dependent, although a low concentration of MgCl