Mutational analysis of the archaeal tyrosine recombinase SSV1 integrase suggests a mechanism of DNA cleavage in trans.
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).
Letzelter
Claire
C
Laboratoire d'Enzymologie des Acides Nucléiques, Institut de Génétique et Microbiologie, Bātiment 400, Université Paris Sud, Orsay Cedex 91405, France.
Duguet
Michel
M
Serre
Marie-Claude
MC
D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D006801:Humans; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D019843:Archaeal Proteins; D004247:DNA; D019426:Integrases; C456321:SSV1 integrase; D004264:DNA Topoisomerases, Type I