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- Title
Rational design of a chimeric endonuclease targeted to NotI recognition site.
- Authors
Penghua Zhang; Yongming Bao; Lauren Higgins; Shuang-yong Xu
- Abstract
A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of the notIR gene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1–198) with non-specific nuclease activity was fused to the NotI variant D160N with a short linker. The engineered chimeric endonuclease (CH-endonuclease) recognizes NotI sites specifically in the presence of high salt (100–150 mM NaCl) and divalent cations Mg++ or Ca++. In contrast to wild-type NotI, which cuts within its recognition sequence, BmrI198-NotI (D160N) cleaves DNA outside of NotI sites, resulting in deletion of the NotI site and the adjacent sequences. The fusion of the BmrI cleavage domain to cleavage-deficient variants of Type II restriction enzymes to generate novel cleavage sites will provide useful tools for DNA manipulation.
- Subjects
ENDONUCLEASES; MUTAGENESIS; GENES; DNA
- Publication
PEDS: Protein Engineering, Design & Selection, 2007, Vol 20, Issue 10, p497
- ISSN
1741-0126
- Publication type
Article
- DOI
10.1093/protein/gzm049