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Asymmetry in Flp-mediated cleavage
K H Luetke1, B P Zhao, P D Sadowski
1Department of Medical Genetics and Microbiology, University of Toronto, MSB, Toronto, Ontario M5S 1A8, Canada.
Nucleic Acids Research
|October 23, 1997
Summary
Flp recombinase, a site-specific enzyme, covalently binds DNA during cleavage. This binding alters the Flp-DNA complex conformation, potentially enabling strand exchange for genetic recombination.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Flp recombinase belongs to the integrase family of site-specific recombinases.
- Integrase family enzymes perform DNA cleavage via transesterification, involving an active site tyrosine.
- The initial reaction step covalently links the protein to the 3'-phosphoryl DNA terminus.
Purpose of the Study:
- To investigate the conformational changes in the Flp-FRT complex during DNA cleavage and covalent attachment.
- To elucidate the mechanism of Flp-mediated DNA strand cleavage and its relation to strand exchange.
Main Methods:
- Utilized Flp recognition target (FRT) sites with a 5'-bridging phosphorothioate linkage to capture Flp-DNA covalent intermediates.
- Employed dimethyl sulfate (DMS) for methylation protection and interference assays to probe DNA-protein interactions.
- Analyzed changes in adenine residue protection and Flp-DNA contacts at cleavage sites.
Main Results:
- Flp-mediated cleavage resulted in the covalent attachment of Flp to the 3'-phosphoryl DNA terminus.
- Two adenine residues opposite the cleavage site were protected from methylation upon Flp binding.
- Methylation interference studies revealed altered Flp contacts with cleavage sites and surrounding elements.
Conclusions:
- Flp-mediated cleavage and covalent attachment induce conformational changes within the Flp-FRT complex.
- These observed conformational alterations are likely crucial for subsequent Flp-mediated strand exchange activity.
- The findings provide mechanistic insights into the function of Flp recombinase in DNA manipulation.