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Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues
Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction–modification (R–M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable res...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Portland Press Ltd.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494987/ https://www.ncbi.nlm.nih.gov/pubmed/32880391 http://dx.doi.org/10.1042/BSR20201633 |
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author | Kumar, Sumith Bangru, Sushant Kumar, Ritesh Rao, Desirazu N. |
author_facet | Kumar, Sumith Bangru, Sushant Kumar, Ritesh Rao, Desirazu N. |
author_sort | Kumar, Sumith |
collection | PubMed |
description | Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction–modification (R–M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni(2+) over Mg(2+). Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes. |
format | Online Article Text |
id | pubmed-7494987 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Portland Press Ltd. |
record_format | MEDLINE/PubMed |
spelling | pubmed-74949872020-09-24 Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues Kumar, Sumith Bangru, Sushant Kumar, Ritesh Rao, Desirazu N. Biosci Rep Protein Engineering Helicobacter pylori is a carcinogenic bacterium that is responsible for 5.5% of all human gastric cancers. H. pylori codes for an unusually large number of restriction–modification (R–M) systems and several of them are strain-specific and phase-variable. HpyAII is a novel Type IIs phase-variable restriction endonuclease present in 26695 strain of H. pylori. We show that HpyAII prefers two-site substrates over one-site substrates for maximal cleavage activity. HpyAII is less stringent in metal ion requirement and shows higher cleavage activity with Ni(2+) over Mg(2+). Mutational analysis of the putative residues of the HNH motif of HpyAII confirms that the protein has an active HNH site for the cleavage of DNA. However, mutation of the first Histidine residue of the HNH motif to Alanine does not abolish the enzymatic activity, but instead causes loss of fidelity compared with wildtype HpyAII. Previous studies have shown that mutation of the first Histidine residue of the HNH motif of all other known HNH motif motif-containing enzymes completely abolishes enzymatic activity. We found, in the case of HpyAII, mutation of an active site residue leads to the loss of endonuclease fidelity. The present study provides further insights into the evolution of restriction enzymes. Portland Press Ltd. 2020-09-16 /pmc/articles/PMC7494987/ /pubmed/32880391 http://dx.doi.org/10.1042/BSR20201633 Text en © 2020 The Author(s). https://creativecommons.org/licenses/by/4.0/ This is an open access article published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons Attribution License 4.0 (CC BY). |
spellingShingle | Protein Engineering Kumar, Sumith Bangru, Sushant Kumar, Ritesh Rao, Desirazu N. Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues |
title | Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues |
title_full | Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues |
title_fullStr | Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues |
title_full_unstemmed | Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues |
title_short | Promiscuous DNA cleavage by HpyAII endonuclease is modulated by the HNH catalytic residues |
title_sort | promiscuous dna cleavage by hpyaii endonuclease is modulated by the hnh catalytic residues |
topic | Protein Engineering |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494987/ https://www.ncbi.nlm.nih.gov/pubmed/32880391 http://dx.doi.org/10.1042/BSR20201633 |
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