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

Abstract

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 .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 tran...

Site-Specific Recombination by SSV2 Integrase: Substrate Requirement and Domain Functions

“…Int SSV1 , the prototype of SSV-type integrases, was shown to catalyze both integration and excision reactions, and no additional accessory proteins were apparently required for the excision reaction, compared to those for the integration reaction, in early biochemical studies (8,16). Like other laboratories, we failed to establish in vitro recombination assays for Int SSV1 (17,18; our unpublished results). However, we were able to set up an in vitro Int SSV2 assay that permitted the biochemical analysis of the recombination activity of SSV-type integrases in this study.…”

“…Those researchers found that Int SSV1 employed a cleavage mechanism utilizing the active-site residue Tyr 314 and formed a 3=-phosphoprotein intermediate during the cleavage reaction, as found with bacterial and eukaryal tyrosine recombinases (18). Like Int, Int SSV1 also exhibited type IB topoisomerase activity (17). Recently, the crystal structure of the C-terminal catalytic domain of Int SSV1 was resolved (19,20).…”

“…An 18-bp sequence within the att site was identified as the binding site for Int SSV1 (16). For unknown reasons, however, the in vitro recombination assays for Int SSV1 have not been successfully established in other laboratories (17,18). Therefore, subsequent biochemical studies have been mostly targeting steps during the cycle of Int SSV1 -catalyzed recombination, such as substrate binding and strand cleavage.…”

Archaeal Extrachromosomal Genetic Elements

“…For unknown reasons, however, their in vitro recombination assays of Int SSV1 have not been successfully established in other laboratories (138). Therefore, in their studies, Serre et al employed assays targeting other activities of Int SSV1 (138,139). They showed that Int SSV1 was able to cleave a 19-bp duplex DNA containing the above-mentioned 18-bp sequence, generating a nick on each of the two DNA strands in vitro.…”

“…Furthermore, an 18-bp sequence within the att site was identified as the binding site for Int SSV1 , as revealed by footprinting. For unknown reasons, however, their in vitro recombination assays of Int SSV1 have not been successfully established in other laboratories (138). Therefore, in their studies, Serre et al employed assays targeting other activities of Int SSV1 (138,139).…”