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Antimicrobial Agents and Chemotherapy, September 2002, p. 2735-2746, Vol. 46, No. 9
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.9.2735-2746.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Importance of the Fourth Alpha-Helix within the CAP Homology Domain of Type II Topoisomerase for DNA Cleavage Site Recognition and Quinolone Action

Dirk Strumberg,^1,4* John L. Nitiss,² Jiaowang Dong,² Jerrylaine Walker,² Marc C. Nicklaus,³ Kurt W. Kohn,⁴ Jonathan G. Heddle,^5, Anthony Maxwell,^5, Siegfried Seeber,¹ and Yves Pommier^4*

Laboratories of Molecular Pharmacology,⁴ Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892,³ Department of Internal Medicine and Medical Oncology, West German Cancer Center, University Medical School of Essen, 45122 Essen, Germany,¹ Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105,² Department of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom⁵

Received 9 January 2002/ Returned for modification 12 April 2002/ Accepted 22 May 2002

We report that point mutations causing alteration of the fourth alpha-helix (4-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 4-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 4-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 4-helix are important in determining the activity and possibly the binding of quinolones to the topoisomerase II-DNA complexes.

* Corresponding author. Mailing address for Yves Pommier: Laboratory of Molecular Pharmacology, Bldg. 37, Rm. 4E28, NIH, Bethesda, MD 20892-4255. Phone: (301) 496-5944. Fax: (301) 402-0752. E-mail: pommier{at}nih.gov.

Present addresses: Protein Design Laboratory, Yokohama City University, Yokohama, Kanagawa 230-0045, Japan.

Present address: Department of Biological Chemistry, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom.

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