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Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency
Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is cha...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479162/ https://www.ncbi.nlm.nih.gov/pubmed/22753027 http://dx.doi.org/10.1093/nar/gks635 |
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author | Ukanis, Mindaugas Sapranauskas, Rimantas Lubys, Arvydas |
author_facet | Ukanis, Mindaugas Sapranauskas, Rimantas Lubys, Arvydas |
author_sort | Ukanis, Mindaugas |
collection | PubMed |
description | Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10I(E204Q). Detailed characterization of plasmids from 64 colonies screened from the library of ∼200 000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis. |
format | Online Article Text |
id | pubmed-3479162 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-34791622012-10-24 Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency Ukanis, Mindaugas Sapranauskas, Rimantas Lubys, Arvydas Nucleic Acids Res Methods Online Type II restriction endonucleases (REases) are one of the basic tools of recombinant DNA technology. They also serve as models for elucidation of mechanisms for both site-specific DNA recognition and cleavage by proteins. However, isolation of catalytically active mutants from their libraries is challenging due to the toxicity of REases in the absence of protecting methylation, and techniques explored so far had limited success. Here, we present an improved SOS induction-based approach for in vivo screening of active REases, which we used to isolate a set of active variants of the catalytic mutant, Cfr10I(E204Q). Detailed characterization of plasmids from 64 colonies screened from the library of ∼200 000 transformants revealed 29 variants of cfr10IR gene at the level of nucleotide sequence and 15 variants at the level of amino acid sequence, all of which were able to induce SOS response. Specific activity measurements of affinity-purified mutants revealed >200-fold variance among them, ranging from 100% (wild-type isolates) to 0.5% (S188C mutant), suggesting that the technique is equally suited for screening of mutants possessing high or low activity and confirming that it may be applied for identification of residues playing a role in catalysis. Oxford University Press 2012-10 2012-06-28 /pmc/articles/PMC3479162/ /pubmed/22753027 http://dx.doi.org/10.1093/nar/gks635 Text en © The Author(s) 2012. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Online Ukanis, Mindaugas Sapranauskas, Rimantas Lubys, Arvydas Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency |
title | Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency |
title_full | Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency |
title_fullStr | Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency |
title_full_unstemmed | Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency |
title_short | Screening for catalytically active Type II restriction endonucleases using segregation-induced methylation deficiency |
title_sort | screening for catalytically active type ii restriction endonucleases using segregation-induced methylation deficiency |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3479162/ https://www.ncbi.nlm.nih.gov/pubmed/22753027 http://dx.doi.org/10.1093/nar/gks635 |
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