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Isolation and characterization of intermediates in site-specific recombination
R Hoess1, A Wierzbicki, K Abremski
1Experimental Station, E.I. Dupont de Nemours & Co., Wilmington, DE 19898.
Summary
Mutant Cre proteins reveal a Holliday junction intermediate in DNA recombination. Some mutants, unable to complete recombination, accumulate this intermediate, suggesting defects prior to its formation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Cre recombinase from bacteriophage P1 mediates site-specific DNA recombination at lox sites.
- Wild-type Cre-lox recombination is a highly concerted process, making intermediates difficult to detect.
Purpose of the Study:
- To investigate the mechanism of Cre-lox recombination by characterizing reaction intermediates.
- To identify and analyze intermediates formed by mutant Cre proteins.
Main Methods:
- Isolation and characterization of mutant Cre proteins.
- Analysis of DNA recombination products and intermediates.
- Biochemical assays to study Cre protein activity in vitro.
Main Results:
- Several Cre mutants accumulate a Holliday structure intermediate during recombination.
- This Holliday structure involves the exchange of one set of DNA strands between recombining partners.
- Wild-type Cre can process the Holliday structure, confirming its role as an intermediate.
- Certain Cre mutants defective in recombination can resolve the Holliday structure.
Conclusions:
- The Holliday structure is a key intermediate in the Cre-lox recombination pathway.
- Mutant Cre proteins provide insights into the stepwise nature of the recombination process.
- Some Cre mutants are impaired in steps preceding Holliday structure formation, while others can resolve it.