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Site-specific recombinase, R, encoded by yeast plasmid pSR1
H Araki1, N Nakanishi, B R Evans
1Department of Biotechnology, Faculty of Engineering, Osaka University, Japan.
Journal of Molecular Biology
|May 5, 1992
Summary
The R protein from Zygosaccharomyces rouxii plasmid pSR1 mediates site-specific DNA recombination in vitro. This process occurs without energy, resulting in inversion or deletion based on DNA site orientation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Zygosaccharomyces rouxii plasmid pSR1 contains inverted repeats with a specific recombination site.
- Site-specific recombination is crucial for genetic element dynamics.
Purpose of the Study:
- To characterize the R gene product (R protein) from Z. rouxii plasmid pSR1.
- To investigate the mechanism of R protein-mediated site-specific recombination in vitro.
Main Methods:
- Production and partial purification of R protein in Escherichia coli.
- In vitro recombination assays using circular and linear DNA substrates.
- Analysis of R protein binding sites and DNA cleavage activity.
Main Results:
- R protein catalyzes site-specific recombination in vitro without an energy source.
- Recombination leads to intramolecular inversion or deletion depending on substrate orientation.
- Circular DNA is a preferred substrate over linear DNA for R-mediated recombination.
- R protein binds to specific 12 bp elements within the plasmid's inverted repeats.
- R protein mediates staggered strand cleavage at the recombination site junction.
Conclusions:
- R protein is the sole protein required for pSR1 site-specific recombination.
- The mechanism involves binding to specific DNA elements and mediating strand cleavage.
- Understanding this recombination system provides insights into plasmid biology and genetic engineering tools.