Structure and function of the shufflon in plasmid R64

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Site-specific recombination generates biological diversity in prokaryotes.
• Plasmid R64 utilizes a unique shufflon system with seven recombination sites (sfx) and a recombinase (Rci).

Purpose of the Study:

• To investigate the mechanism of shufflon multiple inversion system in plasmid R64.
• To elucidate the role of Rci recombinase and sfx site asymmetry in DNA inversion.

Main Methods:

• Overexpression and purification of Rci protein.
• In vitro recombination assays using R64 shufflon system.
• Analysis of sfx sequence elements and their impact on inversion frequency.

Main Results:

• Shufflon inversions create diverse pilV genes, determining recipient specificity in bacterial matings.
• Rci mediates recombination via a 5' protruding 7-bp staggered cut in asymmetric sfx sites.
• Symmetric sfx sequences and altered Rci binding enhance inversion frequency.

Conclusions:

• The asymmetry of sfx sites and Rci's binding properties regulate shufflon recombination.
• The Rci C-terminal domain is crucial for specific binding to asymmetric sfx sites.
• Shufflon system provides a model for understanding DNA inversion and biological diversity.