Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities
| Autor: | Ewa Ciszak, Sophie Laget, Yongming Bao, Shuang-yong Xu, Siu-Hong Chan |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
Recombinant Fusion Proteins
Molecular Sequence Data Protein Engineering Cleavage (embryo) Substrate Specificity 03 medical and health sciences Endonuclease chemistry.chemical_compound Recognition sequence Catalytic Domain Cleave Enzyme Stability Genetics Magnesium Amino Acid Sequence Flap endonuclease Deoxyribonucleases Type II Site-Specific 030304 developmental biology 0303 health sciences Base Sequence biology Nucleic Acid Enzymes Osmolar Concentration 030302 biochemistry & molecular biology Temperature DNA DNA-Binding Proteins Kinetics DNA/RNA non-specific endonuclease Restriction enzyme Biochemistry chemistry biology.protein Sequence Alignment |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkm665 |
| Popis: | Creating endonucleases with novel sequence specificities provides more possibilities to manipulate DNA. We have created a chimeric endonuclease (CH-endonuclease) consisting of the DNA cleavage domain of BmrI restriction endonuclease and C.BclI, a controller protein of the BclI restriction-modification system. The purified chimeric endonuclease, BmrI198-C.BclI, cleaves DNA at specific sites in the vicinity of the recognition sequence of C.BclI. Double-strand (ds) breaks were observed at two sites: 8 bp upstream and 18 bp within the C-box sequence. Using DNA substrates with deletions of C-box sequence, we show that the chimeric endonuclease requires the 5' half of the C box only for specific cleavage. A schematic model is proposed for the mode of protein-DNA binding and DNA cleavage. The present study demonstrates that the BmrI cleavage domain can be used to create combinatorial endonucleases that cleave DNA at specific sequences dictated by the DNA-binding partner. The resulting endonucleases will be useful in vitro and in vivo to create ds breaks at specific sites and generate deletions. |
| Databáze: | OpenAIRE |
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