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).
Highlights
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]
We found that IntSSV has a type IB topoisomerase activity, a property shared by some other tyrosine recombinases (28 –31)
Mutagenesis Strategy and Production of IntSSV Mutants within the Conserved Motifs of Tyrosine Recombinases—The signature of tyrosine recombinases is the conservation of two short regions of similarity in their C-terminal part [1, 2, 4, 5]
Summary
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]. 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 shared active site model may be extended to an archaeal tyrosine recombinase
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