Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities

New England Biolabs, Inc. 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2007; 35(18):6238-48. DOI: 10.1093/nar/gkm665
Source: PubMed


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.

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Available from: Shuang-yong Xu, Jan 21, 2014
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    • "Type IIS restriction endonucleases (REases) such as FokI and BmrI, which have a specific DNA binding domain and a nonspecific DNA cleavage domain separately, are useful to create new sequence-specific endonucleases [18,27]. For this purpose, DNA binding domains such as the Z-conformation-specific Za domain of human RNA adenosine deaminase, Gal4 and zinc finger motifs are fused with the FokI DNA cleavage domain (FN). "
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    • "This study adds to the large number of designed chimeric endonucleases engineered by linking the catalytic domain of FokI (25–37) or the catalytic domain of BmrI (75–77), another type IIS endonuclease, to unrelated DNA-binding domains. What distinguishes our most successful engineered chimeric endonucleases, CdnDI and CdnDII, from their predecessors is that they each make a single, double-stranded break in DNA at a precisely defined site outside their recognition region, in a manner similar to that of type IIS restriction endonucleases. "
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