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Specific contacts between the FLP protein of the yeast 2-micron plasmid and its recombination site
Abstract:
Contact points between the FLP protein of the yeast 2-micron plasmid and its recombination site have been defined. Important features of the region previously defined as the minimal recombination site in vitro include a pair of 13-base pair inverted repeats separated by an 8-base pair spacer. The two FLP protein-binding sites within this region are 12 base pairs in length. In each case they include the internal 11 base pairs of one of the 13-base pair repeats, as well as the adjacent base pair within the spacer. The internal 6 base pairs within the spacer are not involved in binding or recognition by FLP protein. When the size of the spacer is increased or decreased by one base pair, the distance between the cleavage points is also increased or decreased correspondingly by one base pair. Points of cleavage are unaffected by changes in the spacer sequence. Specific contact points involving purine residues, identified by methylation protection and recombination interference experiments, are located in both the major and minor grooves of the DNA. Additional contact points between FLP protein and phosphate groups in the phosphate-deoxyribose backbone are clustered near the cleavage sites.
Insights
The FLP recombinase protein binds specific DNA sequences at yeast 2-micron plasmid recombination sites. Key binding and cleavage sites were identified within inverted repeats and the spacer region.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The yeast 2-micron plasmid utilizes FLP recombinase for site-specific recombination.
- Understanding FLP protein-DNA interactions is crucial for dissecting recombination mechanisms.
Purpose of the Study:
- To define the precise contact points between FLP protein and its recombination site.
- To elucidate the role of the spacer sequence and DNA grooves in FLP binding and cleavage.
Main Methods:
- Methylation protection assays to identify DNA contact points.
- Recombination interference experiments to map protein-DNA interactions.
- In vitro analysis of recombination site variants.
Main Results:
- The FLP binding sites are 12 base pairs, encompassing part of the inverted repeats and the spacer.
- The spacer sequence length, but not its composition, affects cleavage site distance.
- Specific purine contacts occur in both major and minor DNA grooves.
- Phosphate backbone contacts are located near cleavage sites.
Conclusions:
- Detailed map of FLP protein-DNA interactions within the recombination site.
- Spacer length is a critical determinant of FLP-mediated cleavage site positioning.
- FLP protein recognizes DNA in both major and minor grooves, involving purine residues and the phosphate backbone.