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J. Biol. Chem., Vol. 280, Issue 14, 14252-14263, April 8, 2005

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Novel Symmetric and Asymmetric DNA Scission Determinants for Streptococcus pneumoniae Topoisomerase IV and Gyrase Are Clustered at the DNA Breakage Site^*

Elisabetta Leo, Katherine A. Gould, Xiao-Su Pan, Giovanni Capranico¶, Mark R. Sanderson||, Manlio Palumbo**, and L. Mark Fisher

From the Molecular Genetics Group, Department of Basic Medical Sciences-Biochemistry and Immunology, St. George'sHospital Medical School, University of London, London SW17 0RE, United Kingdom, G. Moruzzi Department of Biochemistry, University of Bologna, 40126 Bologna, Italy, ^||Randall Centre for Cell and Molecular Biophysics, King's College, Guys' Campus, London Bridge, London SE1 1UL, United Kingdom, and the ^**Department of Pharmaceutical Sciences, University of Padua, 35131 Padua, Italy

Topoisomerase (topo) IV and gyrase are bacterial type IIA DNA topoisomerases essential for DNA replication and chromosome segregation that act via a transient double-stranded DNA break involving a covalent enzyme-DNA "cleavage complex." Despite their mechanistic importance, the DNA breakage determinants are not understood for any bacterial type II enzyme. We investigated DNA cleavage by Streptococcus pneumoniae topo IV and gyrase stabilized by gemifloxacin and other antipneumococcal fluoroquinolones. Topo IV and gyrase induce distinct but overlapping repertoires of double-strand DNA breakage sites that were essentially identical for seven different quinolones and were augmented (in intensity) by positive or negative supercoiling. Sequence analysis of 180 topo IV and 126 gyrase sites promoted by gemifloxacin on pneumococcal DNA revealed the respective consensus sequences: G(G/C)(A/T)A^*GNNCT(T/A)N(C/A) and GN₄G(G/C)(A/C)G^*GNNCTTN(C/A) (preferred bases are underlined; disfavored bases are in small capitals; N indicates no preference; and asterisk indicates DNA scission between -1 and +1 positions). Both enzymes show strong preferences for bases clustered symmetrically around the DNA scission site, i.e. +1G/+4C, -4G/+8C, and particularly the novel -2A/+6T, but with no preference at +2/+3 within the staggered 4-bp overhang. Asymmetric elements include -3G and several unfavored bases. These cleavage preferences, the first for Gram-positive type IIA topoisomerases, differ markedly from those reported for Escherichia coli topo IV (consensus (A/G)^*T/A) and gyrase, which are based on fewer sites. However, both pneumococcal enzymes cleaved an E. coli gyrase site suggesting overlap in gyrase determinants. We propose a model for the cleavage complex of topo IV/gyrase that accommodates the unique -2A/+6T and other preferences.

Received for publication, January 5, 2005 , and in revised form, January 18, 2005.

^* This work was supported by Project Grant 117/C16747 (to L. M. F. and M. R. S.) from the Biotechnology and Biological Sciences Research Council of the United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Supported by a grant from PRIN 2003, Ministero dell'Istruzione, Universita e Ricerca, Rome, and Bologna University, Italy.

To whom correspondence should be addressed: Molecular Genetics Group, Dept. of Basic Medical Sciences-Biochemistry and Immunology, St. George's Hospital Medical School, University of London, London, SW17 0RE, UK. Tel.: 44-208-725-5782; Fax: 44-208-725-2992; E-mail: lfisher{at}sghms.ac.uk.

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