A new in vitro strand transfer assay for monitoring bacterial class 1 integron recombinase IntI1 activity.
IntI1 integrase is a tyrosine recombinase involved in the mobility of antibiotic resistance gene cassettes within bacterial class 1 integrons. Recent data have shown that its recombination specifically involves the bottom strand of the attC site, but the exact mechanism of the reaction is still unclear. An efficient in vitro assay is still required to better characterize the biochemical properties of the enzyme. In this report we describe for the first time an in vitro system partially reproducing the activity of a recombinant pure IntI1. This new assay, which constitutes the only available in vitro model of recombination by IntI1, was used to determine whether this enzyme might be the sole bacterial protein required for the recombination process. Results show that IntI1 possesses all the features needed for performing recombination between attI and attC sites. However, differences in the in vitro intermolecular recombination efficiencies were found according to the target sites and were correlated with DNA affinities of the enzyme but not with in vivo data. The differential affinity of the enzyme for each site, its capacity to bind to a single-stranded structure at the attC site and the recombination observed with single-stranded substrates unambiguously confirm that it constitutes an important intermediary in the reaction. Our data strongly suggest that the enzyme possesses all the functions for generating and/or recognizing this structure even in the absence of other cellular factors. Furthermore, the in vitro assay reported here constitutes a powerful tool for the analysis of the recombination steps catalyzed by IntI1, its structure-function studies and the search for specific inhibitors.
Dubois
Véronique
V
Laboratory of Cellular and Molecular Microbiology and Pathogenicity (MCMP), UMR 5097-CNRS, University Victor Segalen Bordeaux 2, Bordeaux, France.
Debreyer
Carole
C
Litvak
Simon
S
Quentin
Claudine
C
Parissi
Vincent
V
D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D019426:Integrases / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D011995:Recombination, Genetic; D014734:Vibrio cholerae / Q000201:enzymology*
D017931:DNA Primers; D011994:Recombinant Proteins; D019426:Integrases; C115006:integron integrase IntI1