Isolation and characterization of the Tn3 resolvase synaptic intermediate

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Site-specific recombination is a fundamental process in DNA manipulation.
• Tn3 resolvase mediates a critical DNA recombination event.
• Understanding the mechanism of recombination is key to genetic engineering and molecular biology.

Purpose of the Study:

• To isolate and characterize the synaptic intermediate in Tn3 resolvase-mediated site-specific recombination.
• To elucidate the structural and topological properties of the DNA-resolvase complex (synaptosome).
• To investigate the role of the synaptosome in determining recombination specificity.

Main Methods:

• Isolation and quantitative yield of the synaptic intermediate.
• Characterization using restriction endonuclease mapping.
• Electron microscopy and topological analyses to determine complex structure and DNA supercoiling.
• Stabilization of the intermediate using glutaraldehyde crosslinking.

Main Results:

• The synaptic intermediate (synaptosome) was successfully isolated and characterized.
• The synaptosome is a ~100 Å DNA-resolvase complex formed at specific resolution (res) sites, requiring two supercoiled DNA molecules.
• Synaptosome formation significantly alters DNA supercoiling, protecting -3 supercoils and increasing supercoil density.
• Both direct and inverted res sites can form synaptosomes, indicating orientation is determined within the complex.

Conclusions:

• The synaptosome is a stable intermediate in Tn3 resolvase recombination.
• The formation and structure of the synaptosome play a critical role in the specificity of site-specific recombination.
• A three-stage recognition system involving resolvase binding, synaptosome formation, and intra-complex site orientation determination governs recombination specificity.