Bimodal Recognition of DNA Geometry by Human Topoisomerase IIα: Preferential Relaxation of Positively Supercoiled DNA Requires Elements in the C-Terminal Domain

Human topoisomerase II alpha, but not topoisomerase II beta, can sense the geometry of DNA during relaxation and removes positive supercoils > 10-fold faster than it does negative superhelical twists. In contrast, both isoforms maintain lower levels of DNA cleavage intermediates with positively supercoiled substrates. Since topoisomerase II alpha and II beta differ primarily in their C-terminal domains (CTD), this portion of the protein may play a role in sensing DNA geometry. Therefore, to more fully assess the importance of the topoisomerase II alpha CTD in the recognition of DNA topology, hTop2 alpha Delta 1175, a mutant human enzyme that lacks its CTD, was examined. The mutant enzyme relaxed negative and positive supercoils at similar rates but still maintained lower levels of cleavage complexes with positively supercoiled DNA. Furthermore, when the CTD of topoisomerase II beta was replaced with that of the a isoform, the resulting enzyme. preferentially relaxed positively supercoiled substrates. In contrast, a chimeric topoisomerase II alpha that carried the CTD of the beta isoform lost its ability to recognize the geometry of DNA supercoils during relaxation. These findings demonstrate that human topoisomerase IIa recognizes DNA geometry in a bimodal fashion, with the ability to preferentially relax positive DNA supercoils residing in the CTD. Finally, results with a series of human topoisomerase II alpha mutants suggest that clusters of positively charged amino acid residues in the CTD are required for the enzyme to distinguish supercoil geometry during DNA relaxation and that deletion of even the most C-terminal cluster abrogates this recognition.