Self-association and DNA binding properties of the human topoisomerase IIA α2HTH module

The eukaryotic topoisomerase II is an ubiquitous nuclear enzyme involved in vital cellular functions. It is also the target for some of the most active anticancer drugs. In the various crystal structures of yeast topoisomerase II, the 701–748 segment homologous to the human topoisomerase II α 724–771 segment folds into a compact α2β1α3tα4 conformation, hereafter termed α2HTH module (helix turn helix (HTH), α3tα4). The crystal structure of gyrase A has suggested a model wherein HTH is involved in both the enzyme dimerization and the binding to DNA. These two properties were investigated in solution, using the recombinant α2HTH module of human topoisomerase II α and its synthetic components HTH, α4, α3 and turn. The homology-based structure model of human α2HTH superposed that of yeast in the crystal structure with a rmsd of 1.03 Å. Circular dichroism spectra showed that the helical content of human α2HTH in solution is similar to that of its counterpart within yeast topoisomerase II in the solid state. The chemical cross-linking data indicated that α2HTH self-associated into dimers while gel mobility shift assays and anisotropy fluorescence titrations demonstrated that α2HTH, HTH and α4, but not α3, bind efficiently to DNA (dissociation constants of 3.10–7 M for α2HTH and α4, of 3.10–6 M for HTH and of only 1.10–5 M for α3). Correlatively, α2HTH, α4 and HTH, but not α3, were able to inhibit topoisomerase II in DNA relaxation assays, stipulating that α4 is the DNA recognition helix. All suggests that the α2HTH module once separated from the whole protein conserves a compact conformation, integral to specific dimerization and DNA recognition. The module may thus be used for the search of drugs efficient in hindering topoisomerase II dimerization or binding to DNA.