Cloning and characterization of Drosophila topoisomerase III beta - Relaxation of hypernegatively supercoiled DNA

We cloned cDNA encoding Drosophila DNA topoisomerase III, The top3 cDNA enc odes an 875-amino acid protein, which is nearly 60% identical to mammalian topoisomerase III beta enzymes. Similarity between the Drosophila protein a nd the topoisomerase III beta s is particularly striking in the carboxyl-te rminal region, where all contain eight highly conserved CXXC motifs not fou nd in other topoisomerase III enzymes. We therefore propose the Drosophila protein is a member of the beta-subfamily of topoisomerase III enzymes, The top3 beta gene is a single-copy gene located at 5 E-F on the X chromosome, P-element insertion into the 5'-untranslated region of this gene affects t opoisomerase III beta protein levels, but not the overall fertility and via bility of the fly. We purified topoisomerase III beta to near homogeneity a nd observed relaxation activity only with a hypernegatively supercoiled sub strate, but not with plasmid DNA directly isolated from bacterial cells. De spite this difference in substrate preference, the degree of relaxation of the hypernegatively supercoiled substrate is comparable to relaxation of pl asmid DNA by other type I enzymes. Drosophila topoisomerase III beta forms a covalent linkage to 5' DNA phosphoryl groups, and the DNA cleavage reacti on prefers single-stranded substrate over double-stranded, suggesting an af finity of this enzyme for DNA with non-double-helical structure.