Cloning and Characterization of DrosophilaTopoisomerase IIIÎ

We cloned cDNA encoding Drosophila DNA topoisomerase III. The top3 cDNA encodes an 875-amino acid protein, which is nearly 60% identical to mammalian topoisomerase IIIβ enzymes. Similarity between the Drosophila protein and the topoisomerase IIIβs is particularly striking in the carboxyl-terminal region, where all contain eight highly conserved C XX C motifs not found in other topoisomerase III enzymes. We therefore propose the Drosophila protein is a member of the β-subfamily of topoisomerase III enzymes. The top3 β 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 topoisomerase IIIβ protein levels, but not the overall fertility and viability of the fly. We purified topoisomerase IIIβ to near homogeneity and observed relaxation activity only with a hypernegatively supercoiled substrate, but not with plasmid DNA directly isolated from bacterial cells. Despite this difference in substrate preference, the degree of relaxation of the hypernegatively supercoiled substrate is comparable to relaxation of plasmid DNA by other type I enzymes. Drosophila topoisomerase IIIβ forms a covalent linkage to 5′ DNA phosphoryl groups, and the DNA cleavage reaction prefers single-stranded substrate over double-stranded, suggesting an affinity of this enzyme for DNA with non-double-helical structure.