Cargando…

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...

Descripción completa

Detalles Bibliográficos
Autores principales: Kumar, Sumith, Bangru, Sushant, Kumar, Ritesh, Rao, Desirazu N.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Portland Press Ltd. 2020
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
_version_ 1783582842061586432
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
work_keys_str_mv AT kumarsumith promiscuousdnacleavagebyhpyaiiendonucleaseismodulatedbythehnhcatalyticresidues
AT bangrusushant promiscuousdnacleavagebyhpyaiiendonucleaseismodulatedbythehnhcatalyticresidues
AT kumarritesh promiscuousdnacleavagebyhpyaiiendonucleaseismodulatedbythehnhcatalyticresidues
AT raodesirazun promiscuousdnacleavagebyhpyaiiendonucleaseismodulatedbythehnhcatalyticresidues