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PEDS Advance Access originally published online on October 20, 2007
Protein Engineering Design and Selection 2007 20(10):497-504; doi:10.1093/protein/gzm049
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© 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.

Rational design of a chimeric endonuclease targeted to NotI recognition site

Penghua Zhang, Yongming Bao, Lauren Higgins and Shuang-yong Xu1

New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA

1 To whom correspondence should be addressed. E-mail: xus{at}neb.com

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.

Keywords: BcgI-like enzyme/BmrI/NotI/cleavage-deficient mutant/engineered endonuclease

Received December 8, 2006; revised July 12, 2007; accepted August 9, 2007.


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