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Nucleic Acids Research 2006 34(3):796-805; doi:10.1093/nar/gkj483
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Published online 2 February 2006

© The Author 2006. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants

James C. Samuelson*, Richard D. Morgan, Jack S. Benner, Toby E. Claus, Stephanie L. Packard and Shuang-yong Xu

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

*To whom correspondence should be addressed. Tel: +1 978 380 7288; Fax: +1 978 380 7419; Email: samuelson{at}neb.com

Received October 21, 2005. Revised January 13, 2006. Accepted January 13, 2006.

Restriction endonucleases (REases) with 8-base specificity are rare specimens in nature. NotI from Nocardia otitidis-caviarum (recognition sequence 5'-GCGGCCGC-3') has been cloned, thus allowing for mutagenesis and screening for enzymes with altered 8-base recognition and cleavage activity. Variants possessing altered specificity have been isolated by the application of two genetic methods. In step 1, variant E156K was isolated by its ability to induce DNA-damage in an indicator strain expressing M.EagI (to protect 5'-NCGGCCGN-3' sites). In step 2, the E156K allele was mutagenized with the objective of increasing enzyme activity towards the alternative substrate site: 5'-GCTGCCGC-3'. In this procedure, clones of interest were selected by their ability to eliminate a conditionally toxic substrate vector and induce the SOS response. Thus, specific DNA cleavage was linked to cell survival. The secondary substitutions M91V, F157C and V348M were each found to have a positive effect on specific activity when paired with E156K. For example, variant M91V/E156K cleaves 5'-GCTGCCGC-3' with a specific activity of 8.2 x 104 U/mg, a 32-fold increase over variant E156K. A comprehensive analysis indicates that the cleavage specificity of M91V/E156K is relaxed to a small set of 8 bp substrates while retaining activity towards the NotI sequence.

DDBJ/EMBL/GenBank accession no. DQ242471


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