Expression, domain structure, and enzymatic properties of an active recombinant human DNA topoisomerase IIβ

Abstract

Human cells express two genetically distinct isoforms of DNA topoisomerase II, α and β, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase IIβ in yeast by cloning a topoisomerase IIβ cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase IIβ (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase IIα. Partial digestion of β with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase IIα and β induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug- induced DNA breakage were observed. These studies of human topoisomerase IIβ in concert with α should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.