Recombination by resolvase is inhibited by lac repressor simultaneously binding operators between res sites

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Tn3 resolvase mediates DNA recombination, a process crucial for genetic stability and evolution.
• Efficient recombination by Tn3 resolvase necessitates the alignment of recombination (res) sites as direct repeats.
• Tracking models predict that DNA-tracking proteins must contact DNA between recombination sites.

Purpose of the Study:

• To investigate whether Tn3 resolvase requires direct DNA contact between recombination sites.
• To test the validity of DNA tracking models for Tn3 resolvase activity.
• To elucidate the role of DNA topology and protein interactions in Tn3 resolvase-mediated recombination.

Main Methods:

• Assessing Tn3 resolvase recombination efficiency in the presence of DNA-binding proteins (lac repressor, T7 RNA polymerase).
• Introducing protein diffusion blockades between recombination sites to test tracking models.
• Analyzing the effect of lac repressor binding to multiple operator sites on recombination.

Main Results:

• Recombination was unaffected by proteins bound between the two res sites.
• Recombination was inhibited when lac repressor bound to operators flanking both res sites, forming a DNA loop.
• Lac repressor demonstrated the ability to bind to multiple DNA sites simultaneously under experimental conditions.

Conclusions:

• The findings do not support DNA tracking models for Tn3 resolvase.
• DNA looping and topological constraints imposed by protein binding can inhibit resolvase activity.
• The structure and topology of the DNA substrate are critical for the formation of the synaptic complex required for recombination.