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 C topoisomerase II alpha 724-771 segment folds into a compact alpha(2)beta(1)alpha(3)t alpha(4) conformation, hereafter termed alpha 2HTH module (helix turn helix (HTH), alpha(3)t alpha(4)). 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 alpha(2),HTH module of human topoisomerase II (alpha) and its synthetic components HTH, alpha(4), alpha(3) and turn. The homology-based structure model of human alpha 2HTH superposed that of yeast in the crystal structure with a rmsd of 1.03 angstrom. Circular dichroism spectra showed that the helical content of human alpha 2HTH in solution is similar to that of its counterpart within yeast topoisomerase II in the solid state. The chemical cross-linking data indicated that alpha 2HTH self-associated into dimers while gel mobility shift assays and anisotropy fluorescence titrations demonstrated that alpha 2HTH. HTH and alpha(4). but not a, bind efficiently to DNA (dissociation constants of 3. 10(-7) M for alpha 2HTH and alpha(4), of 3.10(-6) M for HTH and of only 1.10(-5) M for alpha(3). Correlatively, alpha 2HTH, alpha(4) and HTH. but not alpha(3) were able to inhibit topoisomerase II in DNA relaxation assays, stipulating that alpha(4) is the DNA recognition helix. All suggests that the alpha 2HTH 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. (c) 2005 Elsevier SAS. All rights reserved.