Related Experiment Videos
The FLP protein contacts both major and minor grooves of its recognition target sequence
G B Panigrahi1, L G Beatty, P D Sadowski
1Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
Abstract:
The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.
Insights
The FLP recombinase protein binds extensively to both major and minor DNA grooves within the FLP recognition target (FRT) site. This interaction is crucial for yeast 2-micron plasmid DNA recombination, without altering the DNA structure.
Area of Science:
- Molecular Biology
- Yeast Genetics
- DNA Recombination
Background:
- The 2-micron plasmid in Saccharomyces cerevisiae utilizes FLP recombinase for site-specific DNA recombination.
- FLP protein mediates recombination between specific DNA sequences known as FLP recognition target (FRT) sites.
- Understanding the molecular interactions between FLP and FRT is key to elucidating the mechanism of recombination.
Purpose of the Study:
- To investigate the precise binding interactions of the FLP recombinase protein with the FRT DNA sequence.
- To determine which parts of the FRT site are contacted by FLP and whether DNA conformation changes occur upon binding.
Main Methods:
- Chemical probing using dimethyl sulfate, monoacetyl-4-hydroxyaminoquinoline 1-oxide, and potassium permanganate.
- Analysis of major and minor groove DNA contacts.
- Site-specific binding assays with FLP protein and FRT DNA sequences.
Main Results:
- FLP protein exhibits extensive, site-specific interactions with both the major and minor grooves of the FRT DNA.
- These interactions span approximately 10 base pairs within each of the three symmetry elements of the FRT site.
- No evidence was found to suggest that the FRT site adopts a single-stranded conformation upon FLP binding.
Conclusions:
- The FLP-FRT interaction involves significant contacts in both DNA grooves, highlighting a complex binding mechanism.
- The FRT site likely maintains a double-stranded conformation during FLP binding, contrary to potential single-stranded models.
- These findings provide detailed insights into the molecular basis of FLP-mediated site-specific recombination in yeast.