[Show abstract][Hide abstract] ABSTRACT: 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 (Ser740Trp, Gln743Pro, and Thr744Pro) change the selection of type II topoisomerase-mediated DNA cleavage sites promoted by Ca2+ or produced by etoposide, the fluoroquinolone CP-115,953, or mitoxantrone. By contrast, Thr744Ala substitution had minimal effect on Ca2+- 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 Ser83Trp also changed the DNA cleavage pattern generated by Ca2+ or quinolones. Finally, Thr744Pro 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.
Preview · Article · Oct 2002 · Antimicrobial Agents and Chemotherapy