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Originally published In Press as doi:10.1074/jbc.M105505200 on December 7, 2001

J. Biol. Chem., Vol. 277, Issue 5, 3733-3742, February 1, 2002
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Intricate Interactions within the ccd Plasmid Addiction System*

Minh-Hoa Dao-Thi, Daniel CharlierDagger , Remy Loris§, Dominique Maes, Joris Messens, Lode Wyns, and Jan Backmann||

From the Vlaams interuniversitair Instituut voor Biotechnologie, Vrije Universiteit Brussel, Laboratorium voor Ultrastructuur, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium and Dagger  Vrije Universiteit Brussel, Erfelijkheidsleer en Microbiologie, E. Grysonlaan 1, B-1070 Brussel, Belgium

The ccd addiction system plays a crucial role in the stable maintenance of the Escherichia coli F plasmid. It codes for a stable toxin (CcdB) and a less stable antidote (CcdA). Both are expressed at low levels during normal cell growth. Upon plasmid loss, CcdB outlives CcdA and kills the cell by poisoning gyrase. The interactions between CcdB, CcdA, and its promoter DNA were analyzed. In solution, the CcdA-CcdB interaction is complex, leading to various complexes with different stoichiometry. CcdA has two binding sites for CcdB and vice versa, permitting soluble hexamer formation but also causing precipitation, especially at CcdA:CcdB ratios close to one. CcdA alone, but not CcdB, binds to promoter DNA with high on and off rates. The presence of CcdB enhances the affinity and the specificity of CcdA-DNA binding and results in a stable CcdA·CcdB·DNA complex with a CcdA:CcdB ratio of one. This (CcdA2CcdB2)n complex has multiple DNA-binding sites and spirals around the 120-bp promoter region.

* This work was funded by grants from the Onderzoeksraad of the Vrije Universiteit Brussel, the Research Program of the Fund for Scientific Research Flanders (FWO), and the Vlaams interuniversitair Instituut voor Biotechnologie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Research fellow of the FWO.

To whom correspondence should be addressed. Tel.: 32-2-359-02-82; Fax: 32-2-359-02-89; E-mail: ljwyns@vub.ac.be.

|| Present address: International Association for Soaps, Detergents and Maintenance Products, 49 square Marie-Louise, B-1000 Brussels, Belgium.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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