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Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4

Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recogn...

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Autores principales: Belkebir, Abdelkarim, Azeddoug, Houssine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: International Scholarly Research Network 2012
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392985/
https://www.ncbi.nlm.nih.gov/pubmed/25969755
http://dx.doi.org/10.5402/2012/287230
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author Belkebir, Abdelkarim
Azeddoug, Houssine
author_facet Belkebir, Abdelkarim
Azeddoug, Houssine
author_sort Belkebir, Abdelkarim
collection PubMed
description Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg(2+), Mn(2+), and Ca(2+) concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl(2), and 1 mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme.
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spelling pubmed-43929852015-05-12 Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4 Belkebir, Abdelkarim Azeddoug, Houssine ISRN Biochem Research Article Requirement of divalent cations for DNA cleavage is a general feature of type II restriction enzymes with the exception of few members of this group. A new type II restriction endonuclease has been partially purified from Lactococcus lactis KLDS4. The enzyme was denoted as LlaKI and showed to recognize and cleave the same site as FokI. The enzyme displayed a denatured molecular weight of 50 kDa and behaved as a dimer in solution as evidenced by the size exclusion chromatography. To investigate the role of divalent cations in DNA cleavage by LlaKI, digestion reactions were carried out at different Mg(2+), Mn(2+), and Ca(2+) concentrations. Unlike most of type II restriction endonucleases, LlaKI did not require divalent metal ions to cleave DNA and is one of the few metal-independent restriction endonucleases found in bacteria. The enzyme showed near-maximal levels of activity in 10 mM Tris-HCl pH 7.9, 50 mM NaCl, 10 mM MgCl(2), and 1 mM dithiothreitol at 30°C. The presence of DNA modification was also determined and was correlated with the correspondent restriction enzyme. International Scholarly Research Network 2012-09-30 /pmc/articles/PMC4392985/ /pubmed/25969755 http://dx.doi.org/10.5402/2012/287230 Text en Copyright © 2012 A. Belkebir and H. Azeddoug. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Belkebir, Abdelkarim
Azeddoug, Houssine
Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4
title Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4
title_full Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4
title_fullStr Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4
title_full_unstemmed Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4
title_short Characterization of LlaKI, a New Metal Ion-Independent Restriction Endonuclease from Lactococcus lactis KLDS4
title_sort characterization of llaki, a new metal ion-independent restriction endonuclease from lactococcus lactis klds4
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4392985/
https://www.ncbi.nlm.nih.gov/pubmed/25969755
http://dx.doi.org/10.5402/2012/287230
work_keys_str_mv AT belkebirabdelkarim characterizationofllakianewmetalionindependentrestrictionendonucleasefromlactococcuslactisklds4
AT azeddoughoussine characterizationofllakianewmetalionindependentrestrictionendonucleasefromlactococcuslactisklds4