Related Experiment Videos
The isolation of strand-specific nicking endonucleases from a randomized SapI expression library.
James C Samuelson1, Zhenyu Zhu, Shuang-yong Xu
1New England Biolabs, Inc., 32 Tozer Road, Beverly, MA 01915, USA.
Nucleic Acids Research
|July 13, 2004
Summary
Researchers engineered nicking variants of the SapI enzyme by altering its DNA recognition and cleavage mechanism. This study provides a framework for understanding how SapI DNA cleavage occurs.
Area of Science:
- Molecular Biology
- Enzymology
- Biochemistry
Background:
- The Type IIS restriction endonuclease SapI recognizes a specific DNA sequence (5'-GCTCTTC-3') and cleaves DNA strands at defined positions.
- The asymmetric DNA recognition by SapI suggests the potential for creating strand-specific nicking variants.
Purpose of the Study:
- To isolate and characterize nicking variants of the SapI enzyme.
- To investigate the mechanism of SapI DNA cleavage by exploring strand-specific nicking.
Main Methods:
- Utilized in vitro selection procedures on a randomly mutated SapI expression library.
- Designed SapI substrate sites within expression plasmids for selection of nicking variants.
- Employed site- and strand-specific nicking assays to identify enzyme variants.
Main Results:
- Successfully isolated a bottom-strand nicking variant, Nb.SapI-1, with an R420I substitution.
- Identified several top-strand nicking variants, including Q240R, E250K, G271R, and K273R.
- Observed amino acid substitutions suggesting SapI may possess two active sites per monomer.
Conclusions:
- Demonstrated the successful engineering of strand-specific nicking variants from SapI.
- Provided evidence supporting a potential two-active-site model for SapI in DNA cleavage.
- Established a framework for further mechanistic studies of SapI DNA cleavage.