Cleavage properties of an archaeal site-specific recombinase, the SSV1 integrase.
SSV1 is a virus infecting the extremely thermophilic archaeon Sulfolobus shibatae. The viral-encoded integrase is responsible for site-specific integration of SSV1 into its host genome. The recombinant enzyme was expressed in Escherichia coli, purified to homogeneity, and its biochemical properties investigated in vitro. We show that the SSV1 integrase belongs to the tyrosine recombinases family and that Tyr(314) is involved in the formation of a 3'-phosphotyrosine intermediate. The integrase cleaves both strands of a synthetic substrate in a temperature-dependent reaction, the cleavage efficiency increasing with temperature. A discontinuity was observed in the Arrhenius plot above 50 degrees C, suggesting that a conformational transition may occur in the integrase at this temperature. Analysis of cleavage time course suggested that noncovalent binding of the integrase to its substrate is rate-limiting in the cleavage reaction. The cleavage positions were localized on each side of the anticodon loop of the tRNA gene where SSV1 integration takes place. Finally, the SSV1 integrase is able to cut substrates harboring mismatches in the binding site. For the cleavage step, the chemical nature of the base in position -1 of cleavage seems to be more important than its pairing to the opposite strand.
Serre
Marie-Claude
MC
Laboratoire d'Enzymologie et Biochimie Structurales, CNRS Bat. 34, avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France. serre@igmors.u-psud.fr
Letzelter
Claire
C
Garel
Jean-Renaud
JR
Duguet
Michel
M
D001089:Arabinose / Q000378:metabolism; D001105:Archaea / Q000378:metabolism; D020137:Base Pair Mismatch; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D003001:Cloning, Molecular; D003062:Codon; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D019241:Fuselloviridae; D019426:Integrases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019000:Phosphotyrosine / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012343:RNA, Transfer / Q000378:metabolism; D012347:RNA, Transfer, Arg / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D016944:Sulfolobus / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D003062:Codon; D012347:RNA, Transfer, Arg; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D004247:DNA; D012343:RNA, Transfer; D001089:Arabinose; D019426:Integrases; C456321:SSV1 integrase