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Wild-type Flp recombinase cleaves DNA in trans
1Department of Microbiology and Institute of Cell and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
The EMBO Journal
|February 2, 1999
Summary
Site-specific recombinases cleave DNA using a conserved mechanism. This study confirms Flp recombinase exclusively performs DNA cleavage in trans, where separate protein units interact, regardless of experimental conditions.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Site-specific recombinases (SSRs) from the Integrase family share a common chemical mechanism for DNA strand breakage during recombination.
- A conserved Arg-His-Arg (RHR) triad activates the scissile phosphodiester bond, with an active-site tyrosine acting as the nucleophile for DNA cleavage.
Purpose of the Study:
- To resolve conflicting data regarding whether DNA cleavage by Flp recombinase occurs in cis (same monomer) or in trans (separate monomers).
- To determine if Flp recombinase consistently follows a specific cleavage rule (cis or trans) independent of experimental artifacts.
Main Methods:
- Experimental design to test Flp recombinase cleavage mode without using mutant proteins.
- Analysis of DNA cleavage events mediated by wild-type Flp recombinase under specific conditions.
Main Results:
- Experimental results definitively show that Flp recombinase mediates DNA cleavage exclusively in trans.
- This trans cleavage outcome was observed even when the experimental design excluded the use of mutant recombinase proteins.
Conclusions:
- Flp recombinase consistently utilizes a trans cleavage mechanism for DNA strand breakage.
- The findings clarify the cleavage mechanism of Flp recombinase, contributing to a better understanding of the Integrase family's recombination process.