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Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain
Strand-specific DNA nicking endonucleases (NEases) typically nick 3–7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction...
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2022
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8845596/ https://www.ncbi.nlm.nih.gov/pubmed/35178039 http://dx.doi.org/10.3389/fmicb.2021.787073 |
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author | Xu, Shuang-yong |
author_facet | Xu, Shuang-yong |
author_sort | Xu, Shuang-yong |
collection | PubMed |
description | Strand-specific DNA nicking endonucleases (NEases) typically nick 3–7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction. The mutant was fused to a 76-amino acid (aa) DNA nicking domain of phage Gamma HNH (gHNH) NEase. The chimeric enzyme was purified, and it was shown to nick downstream of a composite site 5′ GGATCC-N(4-6)-AC↑CGR 3′ (R, A, or G) or to nick both sides of BamHI site at the composite site 5′ CCG↓GT-N5-GGATCC-N5-AC↑CGG 3′ (the down arrow ↓ indicates the strand shown is nicked; the up arrow↑indicates the bottom strand is nicked). Due to the attenuated activity of the small nicking domain, the fusion nickase is active in the presence of Mn(2+) or Ni(2+), and it has low activity in Mg(2+) buffer. This work provided a proof-of-concept experiment in which a chimeric NEase could be engineered utilizing the binding specificity of a Type II restriction endonucleases (REases) in fusion with a nicking domain to generate infrequent nickase, which bridges the gap between natural REases and homing endonucleases. The engineered chimeric NEase provided a framework for further optimization in molecular diagnostic applications. |
format | Online Article Text |
id | pubmed-8845596 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-88455962022-02-16 Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain Xu, Shuang-yong Front Microbiol Microbiology Strand-specific DNA nicking endonucleases (NEases) typically nick 3–7 bp sites. Our goal is to engineer infrequent NEase with a >8 bp recognition sequence. A BamHI catalytic-deficient mutant D94N/E113K was constructed, purified, and shown to bind and protect the GGATCC site from BamHI restriction. The mutant was fused to a 76-amino acid (aa) DNA nicking domain of phage Gamma HNH (gHNH) NEase. The chimeric enzyme was purified, and it was shown to nick downstream of a composite site 5′ GGATCC-N(4-6)-AC↑CGR 3′ (R, A, or G) or to nick both sides of BamHI site at the composite site 5′ CCG↓GT-N5-GGATCC-N5-AC↑CGG 3′ (the down arrow ↓ indicates the strand shown is nicked; the up arrow↑indicates the bottom strand is nicked). Due to the attenuated activity of the small nicking domain, the fusion nickase is active in the presence of Mn(2+) or Ni(2+), and it has low activity in Mg(2+) buffer. This work provided a proof-of-concept experiment in which a chimeric NEase could be engineered utilizing the binding specificity of a Type II restriction endonucleases (REases) in fusion with a nicking domain to generate infrequent nickase, which bridges the gap between natural REases and homing endonucleases. The engineered chimeric NEase provided a framework for further optimization in molecular diagnostic applications. Frontiers Media S.A. 2022-02-01 /pmc/articles/PMC8845596/ /pubmed/35178039 http://dx.doi.org/10.3389/fmicb.2021.787073 Text en Copyright © 2022 Xu. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Microbiology Xu, Shuang-yong Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain |
title | Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain |
title_full | Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain |
title_fullStr | Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain |
title_full_unstemmed | Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain |
title_short | Engineering Infrequent DNA Nicking Endonuclease by Fusion of a BamHI Cleavage-Deficient Mutant and a DNA Nicking Domain |
title_sort | engineering infrequent dna nicking endonuclease by fusion of a bamhi cleavage-deficient mutant and a dna nicking domain |
topic | Microbiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8845596/ https://www.ncbi.nlm.nih.gov/pubmed/35178039 http://dx.doi.org/10.3389/fmicb.2021.787073 |
work_keys_str_mv | AT xushuangyong engineeringinfrequentdnanickingendonucleasebyfusionofabamhicleavagedeficientmutantandadnanickingdomain |