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J. Biol. Chem., Vol. 280, Issue 5, 3365-3375, February 4, 2005

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The Role of RuvA Octamerization for RuvAB Function in Vitro and in Vivo^*

Cyril V. Privezentzev, Anthony Keeley, Barbara Sigala, and Irina R. Tsaneva

From the Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom

RuvA plays an essential role in branch migration of the Holliday junction by RuvAB as part of the RuvABC pathway for processing Holliday junctions in Escherichia coli. Two types of RuvA-Holliday junction complexes have been characterized: 1) complex I containing a single RuvA tetramer and 2) complex II in which the junction is sandwiched between two RuvA tetramers. The functional differences between the two forms are still not clear. To investigate the role of RuvA octamerization, we introduced three amino acid substitutions designed to disrupt the E. coli RuvA tetramer-tetramer interface as identified by structural studies. The mutant RuvA was tetrameric and interacted with both RuvB and junction DNA but, as predicted, formed complex I only at protein concentrations up to 500 nM. We present biochemical and surface plasmon resonance evidence for functional and physical interactions of the mutant RuvA with RuvB and RuvC on synthetic junctions. The mutant RuvA with RuvB showed DNA helicase activity and could support branch migration of synthetic four-way and three-way junctions. However, junction binding and the efficiency of branch migration of four-way junctions were affected. The activity of the RuvA mutant was consistent with a RuvAB complex driven by one RuvB hexamer only and lead us to propose that one RuvA tetramer can only support the activity of one RuvB hexamer. Significantly, the mutant failed to complement the UV sensitivity of E. coli ruvA cells. These results indicate strongly that RuvA octamerization is essential for the full biological activity of RuvABC.

Received for publication, August 12, 2004 , and in revised form, November 15, 2004.

^* This work was funded by the Wellcome Trust (Project Grant 054358 and Senior Research Fellowship 041244). 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.

Present address: Dept. of Biological Sciences, Imperial College London, London SW7 2AZ, United Kingdom.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom. Tel.: 44-20-7679-2235; Fax: 44-20-7679-7193; E-mail: tsaneva{at}biochem.ucl.ac.uk.

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