ArticleNucleic acids research2006
Engineering a rare-cutting restriction enzyme: genetic screening and selection of NotI variants.
Article in Nucleic acids research, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 31 citations in OpenAlex.
- Expression and Purification ofFrontiers in microbiology · 2022Article
- Engineering altered protein-DNA recognition specificity.Nucleic acids research · 2018Review
- Generation of artificial sequence-specific nucleasesChemical science · 2016Article
- Massively parallel characterization of restriction endonucleases.Nucleic acids research · 2013Article
- Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency.Nucleic acids research · 2012Article
- Rational engineering of sequence specificity in R.MwoI restriction endonuclease.Nucleic acids research · 2012Article
- Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity.Nucleic acids research · 2009Article
- The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases.Nucleic acids research · 2008Article
- Structures of the rare-cutting restriction endonuclease NotI reveal a unique metal binding fold involved in DNA binding.Structure (London, England : 1993) · 2008Article
- Expression and purification of BmrI restriction endonuclease and its N-terminal cleavage domain variants.Protein expression and purification · 2008Article
- Rational design of a chimeric endonuclease targeted to NotI recognition site.Protein engineering, design & selection : PEDS · 2007Article
- Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities.Nucleic acids research · 2007Article
Corrections and comments
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Authors and funding
6 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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 10(4) 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.
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