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Engineering a nicking endonuclease N.AlwI by domain swapping
Y Xu1, K D Lunnen, H Kong
1New England Biolabs, 32 Tozertypeoad, Beverly, MA 01915, USA.
Summary
Genetic engineering of enzymes can be challenging. Researchers created a novel nicking enzyme by swapping domains in a restriction enzyme, altering its function from DNA cleavage to DNA nicking.
Area of Science:
- Molecular Biology
- Enzymology
- Protein Engineering
Background:
- Modifying enzyme function via genetic engineering is complex.
- Type IIs restriction enzymes, like AlwI, possess DNA-binding and catalytic/dimerization domains.
- AlwI functions as a dimer to cleave both DNA strands.
Purpose of the Study:
- To investigate if altering an enzyme's oligomerization state can change its function.
- To engineer a novel nicking enzyme by domain swapping.
Main Methods:
- Domain swapping: The dimerization domain of AlwI was replaced with a nonfunctional domain from N.BstNBI.
- Characterization of the chimeric enzyme (N.AlwI) to assess its oligomerization state and enzymatic activity.
Main Results:
- The chimeric enzyme, N.AlwI, exists as a monomer instead of a dimer.
- Monomeric N.AlwI recognizes the same DNA sequence as AlwI but nicks only the top strand.
- The engineered nicking enzyme exhibits activity comparable to the wild-type AlwI.
Conclusions:
- Altering the oligomerization state of an enzyme through domain swapping can successfully change its function.
- This study reports the first instance of creating a nicking enzyme via domain swapping.
- The engineered monomeric nicking enzyme is a functional and potentially useful tool in molecular biology.