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 (α4-helix) of the CAP homology domain of eukaryotic ( Saccharomyces cerevisiae ) type II topoisomerases (Ser 740 Trp, Gln 743 Pro, and Thr 744 Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca 2+ or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr 744 Ala substitution had minimal effect on Ca 2+ - and drug-stimulated DNA cleavage sites, indicating the selectivity of single amino acid substitutions within the α4-helix on type II topoisomerase-mediated DNA cleavage. The equivalent mutation in the gene for Escherichia coli gyrase causing Ser 83 Trp also changed the DNA cleavage pattern generated by Ca 2+ or quinolones. Finally, Thr 744 Pro 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.