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DNA excision by the Sfi I restriction endonuclease
T J Nobbs1, M D Szczelkun, L M Wentzell
1Department of Biochemistry, School of Medical Sciences, Bristol, BS8 1TD, UK.
Journal of Molecular Biology
|August 12, 1998
Summary
The SfiI endonuclease precisely excises DNA by cleaving four phosphodiester bonds sequentially. This tetrameric enzyme
Area of Science:
- Molecular Biology
- Enzymology
Background:
- SfiI endonuclease is a tetrameric protein.
- It binds to two recognition sites before cleaving DNA strands.
Purpose of the Study:
- Elucidate the precise DNA excision mechanism of SfiI.
- Analyze individual reaction steps of SfiI endonuclease.
Main Methods:
- Analysis of SfiI reactions on a plasmid with two SfiI sites.
- Equilibrium centrifugation to confirm SfiI structure.
Main Results:
- Concerted cleavage of four phosphodiester bonds occurs via four consecutive reactions.
- Each reaction has similar intrinsic rate constants, mediated by active sites.
- Complete cleavage occurs within one minute at physiological temperature, but dissociation takes an hour.
Conclusions:
- SfiI mediates precise DNA excision through sequential phosphodiester bond cleavage.
- The tetrameric structure facilitates the concerted action of four active sites.
- Enzyme-product complex dissociation rate limits the overall DNA excision process.