Related Experiment Videos
Point mutations in the integron integrase IntI1 that affect recombination and/or substrate recognition
1Centre 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.
Journal of Bacteriology
|October 10, 1998
Summary
Mutations in conserved residues of integron integrase IntI1 affect its function. While some mutants retain DNA binding, only specific arginine mutations allow for cassette excision, though with reduced efficiency.
Area of Science:
- Molecular Biology
- Microbiology
- Biochemistry
Background:
- Class 1 integrons utilize the site-specific recombinase IntI1 to facilitate the movement of gene cassettes, frequently conferring antibiotic resistance.
- IntI1 belongs to the tyrosine recombinase family, characterized by conserved residues in box I and box II regions, including two arginines and one tyrosine.
Purpose of the Study:
- To investigate the functional roles of three invariant residues within IntI1 by analyzing mutant proteins.
- To assess the impact of point mutations on IntI1's in vivo recombination activity and in vitro DNA binding capabilities.
Main Methods:
- Generation of IntI1 variants with point mutations at conserved arginine (R146, R280) and tyrosine (Y312) residues.
- Evaluation of in vitro DNA binding to the attI1 site using mutant proteins.
- Assessment of in vivo cassette excision activity for the generated IntI1 mutants.
Main Results:
- Mutations at R146 and R280 (MBP-IntI1(R146K), MBP-IntI1(R280K)) allowed for in vitro binding to the attI1 site.
- Only the MBP-IntI1(R280K) mutant demonstrated in vivo cassette excision, albeit with lower efficiency than wild-type IntI1.
- Mutations at the conserved tyrosine residue (Y312) did not impair DNA binding but abolished in vivo recombination activity.
Conclusions:
- The conserved arginine residues in IntI1 are crucial for both DNA binding and catalytic activity in recombination.
- The conserved tyrosine residue is essential for the in vivo catalytic function of IntI1, independent of DNA binding.
- Understanding these residues' roles provides insights into the mechanism of site-specific recombination mediated by integron integrases.