Point mutations in the integron integrase IntI1 that affect recombination and/or substrate recognition.
The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system. The integron integrase belongs to the tyrosine recombinase (phage integrase) family. The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II. Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II. We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding. We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo. However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1. We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.
Centre de Recherche en Infectiologie, Centre Hospitalier de l'Université Laval and Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Sainte-Foy, Québec, Canada.
A
A
Gravel
N
N
Messier
P H
PH
Roy
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D005810:Multigene Family; D017354:Point Mutation; D011485:Protein Binding; D011995:Recombination, Genetic; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D019426:Integrases; C115006:integron integrase IntI1