Importance of the fourth alpha-helix within the CAP homology domain of type II topoisomerase for DNA cleavage site recognition and quinolone action

We report that point mutations causing alteration of the fourth alpha-helix (α14-helix) of the CAP homology domain of eukaryotic (Saccharomyces cerevisiae) type II topoisomerases (SerTrp, GlnPro, and ThrPro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, ThrAla substitution had minimal effect on Ca- and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the α14-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing SerTrp also changed the DNA cleavage pattern generated by Ca or quinolones. Finally, ThrPro substitution in the yeast type II topoisomerase rendered the enzyme sensitive to antibacterial quinolones. This study shows that the α4-helix within the conserved CAP homology domain of type II topoisomerases is critical for selecting the sites of DNA cleavage. It also demonstrates that selective amino acid residues in the α4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.