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Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity
The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition speci...
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Formato: | Texto |
Lenguaje: | English |
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Oxford University Press
2009
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731914/ https://www.ncbi.nlm.nih.gov/pubmed/19567736 http://dx.doi.org/10.1093/nar/gkp535 |
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author | Morgan, Richard D. Luyten, Yvette A. |
author_facet | Morgan, Richard D. Luyten, Yvette A. |
author_sort | Morgan, Richard D. |
collection | PubMed |
description | The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature. |
format | Text |
id | pubmed-2731914 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-27319142009-09-10 Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity Morgan, Richard D. Luyten, Yvette A. Nucleic Acids Res Nucleic Acid Enzymes The type II restriction endonucleases are indispensible tools for molecular biology. Although enzymes recognizing nearly 300 unique sequences are known, the ability to engineer enzymes to recognize any sequence of choice would be valuable. However, previous attempts to engineer new recognition specificity have met limited success. Here we report the rational engineering of multiple new type II specificities. We recently identified a family of MmeI-like type II endonucleases that have highly similar protein sequences but different recognition specificity. We identified the amino-acid positions within these enzymes that determine position specific DNA base recognition at three positions within their recognition sequences through correlations between their aligned amino-acid residues and aligned recognition sequences. We then altered the amino acids at the identified positions to those correlated with recognition of a desired new base to create enzymes that recognize and cut at predictable new DNA sequences. The enzymes so altered have similar levels of endonuclease activity compared to the wild-type enzymes. Using simple and predictable mutagenesis in this family it is now possible to create hundreds of unique new type II restriction endonuclease specificities. The findings suggest a simple mechanism for the evolution of new DNA specificity in Nature. Oxford University Press 2009-08 2009-06-30 /pmc/articles/PMC2731914/ /pubmed/19567736 http://dx.doi.org/10.1093/nar/gkp535 Text en © 2009 The Author(s) http://creativecommons.org/licenses/by-nc/2.0/uk/ 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. |
spellingShingle | Nucleic Acid Enzymes Morgan, Richard D. Luyten, Yvette A. Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity |
title | Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity |
title_full | Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity |
title_fullStr | Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity |
title_full_unstemmed | Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity |
title_short | Rational engineering of type II restriction endonuclease DNA binding and cleavage specificity |
title_sort | rational engineering of type ii restriction endonuclease dna binding and cleavage specificity |
topic | Nucleic Acid Enzymes |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2731914/ https://www.ncbi.nlm.nih.gov/pubmed/19567736 http://dx.doi.org/10.1093/nar/gkp535 |
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