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Updated: Jun 27, 2026
09:19
Analysis of SNARE-mediated Membrane Fusion Using an Enzymatic Cell Fusion Assay
Published on: October 19, 2012
Synapsis and catalysis by activated Tn3 resolvase mutants
Femi J Olorunniji1, Jiuya He, Sandra V C T Wenwieser
1Faculty of Biomedical & Life Sciences, University of Glasgow, Glasgow, Scotland, UK.
Nucleic Acids Research
|November 19, 2008
Summary
Activated serine recombinase variants efficiently catalyze DNA recombination at specific sites. These mutants form stable complexes, revealing new insights into the mechanisms of DNA synapsis and regulation in recombination.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- The serine recombinase Tn3 resolvase mediates recombination between specific DNA sequences called res sites.
- Each res site contains binding sites for multiple resolvase dimers, crucial for wild-type recombination.
- Understanding the precise mechanism of synapsis and regulation is key to comprehending Tn3 resolvase function.
Purpose of the Study:
- To investigate the in vitro properties of Tn3 resolvase variants with activating mutations.
- To elucidate the mechanism of synapse formation and DNA cleavage in these activated variants.
- To compare the activity and properties of activated variants with wild-type Tn3 resolvase.
Main Methods:
- In vitro analysis of purified Tn3 resolvase variants with activating mutations.
- Characterization of recombination activity at specific DNA binding sites (site I).
- Analysis of DNA-protein complex formation (synapse) using biochemical assays.
Main Results:
- Activated resolvase variants can catalyze recombination at a single binding site (site I) efficiently, comparable to wild-type recombination at full res sites.
- These variants exhibit reduced topological selectivity and do not require the subunit interface essential for wild-type activity.
- Activated mutants form stable synapses with a resolvase tetramer and two site I DNA molecules, with DNA cleavage occurring after synapsis.
Conclusions:
- The synapse is assembled through sequential binding of resolvase monomers to site I, followed by the interaction of two site I-dimer complexes.
- Activating mutations alter the mechanism of synapsis and regulation, providing insights into wild-type resolvase function.
- These findings contribute to a deeper understanding of serine recombinase mechanisms and DNA recombination regulation.
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