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

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... tant 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 . Tyrosine recombinases form a large family of site-specific recombinases comprising more than 150 members, most of which were identified on the basis of sequence similarities (1, 2). Within this family, several subfamilies can be defined such as the -phage integrase family, the Xer recombinases family, or the yeast plasmid recombinases family (1). The hallmark of tyrosine recombinases is the conservation of six noncontiguous residues: Arg I , Lys ␤ , His II , Arg II , His/Trp, Tyr (Table I) . This motif is directly involved in catalysis of DNA strand cleavage and strand exchange (for review, see Ref. 3). Five of the six residues are located within the highly conserved boxes I and II found in tyrosine recombinases (1, 4, 5), whereas the sixth residue, Lys ␤ , was identified by alignments with the eukaryotic topoisomerases IB (6). Two different structural organizations of this motif have been described from crystallographic data. In prokaryotic tyrosine recombinases XerD (7), Cre (8), HP1 integrase (9), and -Int (10, 11), the six active site residues come from a single monomer, whereas the eukaryotic Flp recombinase presents a shared active site, where the catalytic tyrosine is provided by one monomer, and the five other residues are from another monomer (12). In this latter case, the active site is created by dimer association. As a consequence of this organization, Flp realizes trans cleavage (13, 14) , whereas prokaryotic recombinases act in cis (8, 9, (15) (16) (17) . Cis cleavage is the result of cis activation/cis cleavage where the tyrosine of the bound monomer attacks the nearby activated phosphate. In trans cleavage, binding of a monomer to its site leads to activation of the adjacent phosphodiester that will be attacked by a nucleophile (here a tyrosine) provided in trans by a partner monomer, a mechanism that can be described as cis activation/ trans cleavage. In both cases the chemistry of the reaction is conserved and is similar to that used by topoisomerases IB (18). The Arg I , His II , and Arg II side chains coordinate the scissile phosphate, activating it for nucleophilic attack by the tyrosine and stabilizing the resulting transient penta-coordinated phosphate. In some proteins, the His/Trp side chain forms a hydrogen bond to the nonbridging oxygen of the scissile phosphate (19), whereas in Flp this residue is more likely involved in protein-protein interactions (20). The Lys ␤ residue is critical for activity of topoisomerases IB and tyrosine recombinases (6, 21, 22) . Enzymatic analysis of vaccinia topoisomerase IB mutants revealed that this residue is the general acid catalyst that protonates the 5Ј-oxygen of the leaving strand (21). Crystal structures of Cre (8), Flp (12), -Int (10), and human topoisomerase IB (19) reveal that this residue contacts the base adjacent to the cleavage site in the minor groove. Other structural data show that Lys ␤ is located on a loop displaying a high conformational flexibility (9, 23). Therefore the Lys ␤ residue was proposed to serve similarly as a general acid in the reaction mechanism catalyzed by tyrosine recombinases (21). So far, the only studied archaeal member of the tyrosine recombinases family is the SSV1 integrase (Int SSV ) 1 encoded by SSV1, a virus of the extremely thermophilic archaeon Sulfolobus shibatae. Int SSV catalyzes the site-specific integration of the viral DNA into the host chromosome using viral and chromosomal attachment sites attP and attB (24, 25) . In a previous work we have shown that Int SSV exhibits a cleavage mechanism dependent on Tyr 314 , leading to the formation of a 3Јphosphoprotein intermediate like other tyrosine recombinases (26). However, Int SSV harbors substitutions at several conserved positions ( Fig. 1 and Ref. 2) and cannot be classified phylogenetically in any subgroup of the tyrosine recombinases family. Whether the recombination reaction catalyzed by Int SSV would follow the general mechanism described for tyrosine recombinases or would be different in archaea remained an open question (27). To characterize further the site-specific recombination mechanism in archaea we have generated Int SSV mutants and analyzed their enzymatic properties. Eight