Nucleic Acids Research Advance Access originally published online on September 13, 2007
Nucleic Acids Research 2007 35(18):6238-6248; doi:10.1093/nar/gkm665
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Nucleic Acids Research, 2007, Vol. 35, No. 18 6238-6248
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
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
*To whom correspondence should be addressed. Tel: +1 978 380 7287; Fax: +1 978 921 1350; Email: xus{at}neb.com
Received December 14, 2006. Revised August 12, 2007. Accepted August 12, 2007.
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.
Present address: Yongming Bao, Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, 116024, P. R. China.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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