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The FLP recombinase of the yeast 2-micron plasmid: characterization of its recombination site
Abstract:
The minimal size of the recombination site required for efficient FLP recombinase-catalyzed recombination in vitro is no more than 28 base pairs, which includes parts of two 13-base-pair inverted repeats and all of an 8-base-pair spacer. The FLP recombinase cleaves the DNA at the boundaries of the spacer, becomes covalently linked to the spacer DNA via a 3' phosphate, and leaves a free 5' hydroxyl at the other end of the 8-base-pair spacer. The efficiency of recombination is reduced if the size of the spacer in a recombinant site is increased or decreased by 1 base pair, while the spacer in the second site is unaltered. Recombination between two sites with identical 1-base-pair additions or deletions in the spacer, however, is relatively unaffected. This result suggests that pairing of sequences in the spacer region is important in FLP-promoted recombination events. The sequence asymmetry utilized by the recombinase to determine the orientation of the site is located uniquely within the spacer region.
Insights
The minimal 28-base-pair recombination site for FLP recombinase requires an 8-base-pair spacer for efficient DNA recombination. Spacer sequence pairing is crucial for FLP-promoted recombination and determining site orientation.
Area of Science:
- Molecular Biology
- Genetics
Background:
- Site-specific recombination is a fundamental biological process.
- FLP recombinase is a widely used tool for genetic engineering and manipulating DNA sequences.
Purpose of the Study:
- To determine the minimal DNA sequence requirements for efficient FLP recombinase-catalyzed recombination in vitro.
- To investigate the role of the spacer region within the recombination site in FLP recombination efficiency and orientation determination.
Main Methods:
- In vitro recombination assays were performed using FLP recombinase and modified recombination sites.
- Variations in the size of the 8-base-pair spacer region were introduced to assess their impact on recombination efficiency.
Main Results:
- The minimal functional recombination site is 28 base pairs, comprising inverted repeats and an 8-base-pair spacer.
- Altering the spacer size by a single base pair significantly reduced recombination efficiency.
- Recombination efficiency was less affected by identical single-base-pair changes in both recombination sites.
- Sequence asymmetry within the spacer dictates the orientation of the recombination site.
Conclusions:
- The 8-base-pair spacer is essential for efficient FLP recombination.
- Spacer sequence pairing plays a critical role in FLP-promoted recombination.
- The spacer region contains the sequence asymmetry necessary for orientation determination by FLP recombinase.