Rational design of a chimeric endonuclease targeted to NotI recognition site
| Autor: | Penghua Zhang, Lauren Higgins, Shuang-yong Xu, Yongming Bao |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Recombinant Fusion Proteins
Molecular Sequence Data Mutant Chimeric Proteins Bioengineering Cleavage (embryo) Biochemistry Article DNA sequencing Substrate Specificity Endonuclease chemistry.chemical_compound Recognition sequence DNA Cleavage Deoxyribonucleases Type II Site-Specific Molecular Biology Gene Sequence Deletion Nuclease Base Sequence biology Temperature Molecular biology Protein Structure Tertiary Restriction enzyme chemistry Drug Design biology.protein Electrophoresis Polyacrylamide Gel DNA Biotechnology |
| Zdroj: | Protein Engineering Design and Selection. 20:497-504 |
| ISSN: | 1741-0134 1741-0126 |
| Popis: | 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-endonu-clease) 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. |
| Databáze: | OpenAIRE |
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