Purification and characterization of human DNA topoisomerase III alpha

Human topoisomerase III alpha (hTopo III alpha), the recently identified fi rst member of the topoisomerase IA subfamily in humans, has a central domai n which is highly homologous to the yeast topoisomerase III, but an overall organization closer to that of Escherichia coli DNA topoisomerase I. In or der to determine the properties of hTopo III alpha, compared to those of ot her topoisomerase IA subfamily members, we purified this enzyme to near hom ogeneity, 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 appea rance of intermediate topoisomers, This DNA relaxation activity is magnesiu m-dependent, although a low concentration of MgCl2 is sufficient to obtain efficient catalysis. P-32-transfer experiments demonstrated that hTopo III alpha is able to cleave a single-stranded oligonucleotide and to bind coval ently to the 5'-end of the cleaved DNA, Addition of 0.5 M NaCl reverses the reaction, leading to the religation of the oligonucleotide, Experiments ut ilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleav age (CANNN down arrow), which is different from that for other members of t he Topo IA subfamily.