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J. Biol. Chem., Vol. 279, Issue 28, 28936-28944, July 9, 2004

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Mutational Analysis of the Archaeal Tyrosine Recombinase SSV1 Integrase Suggests a Mechanism of DNA Cleavage in trans^*

Claire Letzelter, Michel Duguet, and Marie-Claude Serre

From the Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, Btiment 400, Université Paris Sud, Orsay Cedex 91405, France

The only tyrosine recombinase so far studied in archaea, the SSV1 integrase, harbors several changes in the canonical residues forming the catalytic pocket of this family of recombinases. This raised the possibility of a different mechanism for archaeal tyrosine recombinase. The residues of Int^SSV tentatively involved in catalysis were modified by site-directed mutagenesis, and the properties of the corresponding mutants were studied. The results show that all of the targeted residues are important for activity, suggesting that the archaeal integrase uses a mechanism similar to that of bacterial or eukaryotic tyrosine recombinases. In addition, we show that Int^SSV exhibits a type IB topoisomerase activity because it is able to relax both positive and negative supercoils. Interestingly, in vitro complementation experiments between the inactive integrase mutant Y314F and all other inactive mutants restore in all cases enzymatic activity. This suggests that, as for the yeast Flp recombinase, the active site is assembled by the interaction of the tyrosine from one monomer with the other residues from another monomer. The shared active site paradigm of the eukaryotic Flp protein may therefore be extended to the archaeal tyrosine recombinase Int^SSV.

Received for publication, April 9, 2004 , and in revised form, April 26, 2004.

^* This work was supported in part by the CNRS and the Université Paris XI. 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.

Recipient of a fellowship from the Ministère de la Recherche.

To whom correspondence should be addressed. Tel.: 33-1-69-15-6205; Fax: 33-1-69-15-7296; E-mail: serre{at}igmors.u-psud.fr.

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