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Step-arrest mutants of FLP recombinase: implications for the catalytic mechanism of DNA recombination
R L Parsons1, P V Prasad, R M Harshey
1Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Abstract:
The site-specific recombinase (FLP) encoded by the yeast plasmid 2 micron circle belongs to the integrase (of phage lambda) family of recombinases. The sparse homology within the members of this family contrasts with the invariance of three residues, His-396, Arg-399, and Tyr-433 (the numbers correspond to the family alignment positions), among them. We report here results on substrate recognition and catalysis by FLP proteins altered at these residues. Mutations of the conserved His and Tyr that aborted the reaction at specific steps of catalysis permitted genetic dissection of the possible biochemical steps of recombination. We provide indirect evidence that recombination by FLP proceeds through a Holliday junction intermediate.
Insights
Site-specific recombinase FLP (FLP) protein alterations reveal key catalytic steps. Mutations suggest FLP recombination proceeds via a Holliday junction intermediate, advancing understanding of DNA recombination mechanisms.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The FLP recombinase from yeast plasmid 2-micron circle is part of the integrase family.
- Despite low sequence homology, conserved residues His-396, Arg-399, and Tyr-433 are invariant within this family.
Purpose of the Study:
- To investigate the roles of conserved residues (His-396, Arg-399, Tyr-433) in FLP protein function.
- To dissect the biochemical steps of FLP-mediated DNA recombination.
Main Methods:
- Site-directed mutagenesis of conserved residues in FLP recombinase.
- Analysis of substrate recognition and catalytic activity of mutant FLP proteins.
- Genetic dissection of recombination intermediates.
Main Results:
- Mutations at conserved His and Tyr residues disrupted specific catalytic steps.
- These mutations allowed for the genetic dissection of recombination pathways.
- Indirect evidence supports a model where FLP recombination involves a Holliday junction intermediate.
Conclusions:
- Conserved residues His-396 and Tyr-433 are critical for FLP catalysis.
- FLP-mediated recombination likely proceeds through a Holliday junction intermediate.
- Understanding these mechanisms provides insights into site-specific DNA recombination.