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The Cfr10I restriction enzyme is functional as a tetramer
V Siksnys1, R Skirgaila, G Sasnauskas
1Institute of Biotechnology, Lithuania. Siksnys@ibt.lt
Journal of Molecular Biology
|October 16, 1999
Summary
Cfr10I restriction enzyme functions as a homotetramer, not a homodimer. Mutating a key residue confirmed the tetramer is essential for DNA cleavage and interaction with multiple recognition sites.
Area of Science:
- Molecular Biology
- Enzymology
- Structural Biology
Background:
- Most type II restriction endonucleases function as homodimers.
- Cfr10I recognizes the 5'-Pu decreases CCGGPy sequence.
Purpose of the Study:
- To investigate the quaternary structure and function of the Cfr10I restriction enzyme.
- To determine if the crystal structure reflects the solution state of Cfr10I.
Main Methods:
- Gel-filtration and analytical ultracentrifugation to assess Cfr10I oligomerization.
- Site-directed mutagenesis (W220A) to probe the dimer-dimer interface.
- Equilibrium sedimentation and DNA cleavage assays to evaluate mutant function.
- Electron microscopy to visualize DNA-protein interactions.
Main Results:
- Cfr10I exists as a homotetramer in solution.
- The W220A mutation shifts the equilibrium towards a dimer and drastically reduces DNA cleavage activity (<0.1% of wild-type).
- Cfr10I tetramers can bind to two recognition sites on the same DNA molecule, inducing DNA looping.
Conclusions:
- The Cfr10I homotetramer is the functionally relevant form for DNA binding and cleavage.
- The tetrameric structure facilitates interaction with distant recognition sites through DNA looping.