Purification and characterization of human DNA topoisomerase IIIα

Abstract

Human topoisomerase IIIα (hTopo IIIα), 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 IIIα, 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 IIIα 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 MgCl2 is sufficient to obtain efficient catalysis. 32P-transfer experiments demonstrated that hTopo IIIα is able to cleave a single-stranded oligonucleotide and to bind covalently 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 utilizing several different single-stranded oligonucleotides permitted us to map several cleavage sites and to deduce a consensus sequence for DNA cleavage (CANNN↓), which is different from that for other members of the Topo IA subfamily.