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Nucleic Acids Research Advance Access published online on August 22, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn539
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Clerocidin selectively modifies the gyrase-DNA gate to induce irreversible and reversible DNA damage

Xiao Su Pan1, Miriam Dias1, Manlio Palumbo2 and L. Mark Fisher1,*

1Molecular Genetics Group, Molecular and Metabolic Signalling Centre, Division of Basic Medical Sciences, St George's, University of London, Cranmer Terrace, London, SW17 0RE, UK and 2Department of Pharmaceutical Sciences, University of Padua, 35131 Padua, Italy

*To whom correspondence should be addressed. Tel: +44 208 725 5782; Fax: +44 208 725 2992; Email: lfisher{at}sgul.ac.uk

Received July 4, 2008. Revised August 1, 2008. Accepted August 6, 2008.

Clerocidin (CL), a microbial diterpenoid, reacts with DNA via its epoxide group and stimulates DNA cleavage by type II DNA topoisomerases. The molecular basis of CL action is poorly understood. We establish by genetic means that CL targets DNA gyrase in the Gram-positive bacterium Streptococcus pneumoniae, and promotes gyrase-dependent single- and double-stranded DNA cleavage in vitro. CL-stimulated DNA breakage exhibited a strong preference for guanine preceding the scission site (–1 position). Mutagenesis of –1 guanines to A, C or T abrogated CL cleavage at a strong pBR322 site. Surprisingly, for double-strand breaks, scission on one strand consistently involved a modified (piperidine-labile) guanine and was not reversed by heat, salt or EDTA, whereas complementary strand scission occurred at a piperidine-stable –1 nt and was reversed by EDTA. CL did not induce cleavage by a mutant gyrase (GyrA G79A) identified here in CL-resistant pneumococci. Indeed, mutations at G79 and at the neighbouring S81 residue in the GyrA breakage-reunion domain discriminated poisoning by CL from that of antibacterial quinolones. The results suggest a novel mechanism of enzyme inhibition in which the –1 nt at the gyrase-DNA gate exhibit different CL reactivities to produce both irreversible and reversible DNA damage.


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[Abstract] [Full Text] [PDF]


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