Identification of key structural determinants of the IntI1 integron integrase that influence attC x attI1 recombination efficiency.
The integron platform codes for an integrase (IntI) from the tyrosine family of recombinases that mediates recombination between a proximal double-strand recombination site, attI and a single-strand target recombination site, attC. The attI site is only recognized by its cognate integrase, while the various tested attCs sites are recombined by several different IntI integrases. We have developed a genetic system to enrich and select mutants of IntI1 that provide a higher yield of recombination in order to identify key protein structural elements important for attC x attI1 recombination. We isolated mutants with higher activity on wild type and mutant attC sites. Interestingly, three out of four characterized IntI1 mutants selected on different substrates are mutants of the conserved aspartic acid in position 161. The IntI1 model we made based on the VchIntIA 3D structure suggests that substitution at this position, which plays a central role in multimer assembly, can increase or decrease the stability of the complex and accordingly influence the rate of attI x attC recombination versus attC x attC. These results suggest that there is a balance between the specificity of the protein and the protein/protein interactions in the recombination synapse.
Demarre
Gaëlle
G
Unité Plasticité du Génome Bactérien, CNRS URA 2171 and Laboratoire de Biochimie et Biophysique des Macromolécules, CNRS URA 2185, Institut Pasteur, 25 rue du Dr Roux, 75724, Paris 75724, France.
Frumerie
Clara
C
Gopaul
Deshmukh N
DN
Mazel
Didier
D
D001287:Attachment Sites, Microbiological; D001483:Base Sequence; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D015723:Gene Library; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D019426:Integrases; C115006:integron integrase IntI1