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Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis

OBJECTIVE: Planarians including Dugesia ryukyuensis (Dr) have strong regenerative abilities that require enhanced DNA replication. Knockdown of the DUT gene in Dr, which encodes deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase), promotes DNA fragmentation, inhibits regeneration, and eventually...

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Autores principales: Alam, Md. Shahanoor, Moriyama, Hideaki, Matsumoto, Midori
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431053/
https://www.ncbi.nlm.nih.gov/pubmed/30902068
http://dx.doi.org/10.1186/s13104-019-4191-6
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author Alam, Md. Shahanoor
Moriyama, Hideaki
Matsumoto, Midori
author_facet Alam, Md. Shahanoor
Moriyama, Hideaki
Matsumoto, Midori
author_sort Alam, Md. Shahanoor
collection PubMed
description OBJECTIVE: Planarians including Dugesia ryukyuensis (Dr) have strong regenerative abilities that require enhanced DNA replication. Knockdown of the DUT gene in Dr, which encodes deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase), promotes DNA fragmentation, inhibits regeneration, and eventually leads to death. dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. dUTPase is known to prevent uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP, and contributes to genome stability. Nevertheless, the catalytic performance of Dr-dUTPase has not been reported. RESULTS: To confirm the catalytic activity of Dr-dUTPase, we cloned and expressed Dr-DUT in E. coli. Then, we purified Dr-dUTPase using His-tag and removed the tag with thrombin. The resulting Dr-dUTPase had the leading peptide Gly–Ser–His– originating from the vector at the amino terminus, and a mutation, Arg66Lys, to remove the internal thrombin site. We observed the hydrolysis of dUTP by Dr-dUTPase using Cresol Red as a proton sensor. The K(m) for dUTP was determined to be 4.0 µM, which is similar to that for human dUTPase. Dr-dUTPase exhibited a preference for dUTP over the other nucleotides. We conclude the Dr-dUTPase has catalytic activity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13104-019-4191-6) contains supplementary material, which is available to authorized users.
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spelling pubmed-64310532019-04-04 Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis Alam, Md. Shahanoor Moriyama, Hideaki Matsumoto, Midori BMC Res Notes Research Note OBJECTIVE: Planarians including Dugesia ryukyuensis (Dr) have strong regenerative abilities that require enhanced DNA replication. Knockdown of the DUT gene in Dr, which encodes deoxyuridine 5′-triphosphate pyrophosphatase (dUTPase), promotes DNA fragmentation, inhibits regeneration, and eventually leads to death. dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate. dUTPase is known to prevent uracil misincorporation in DNA by balancing the intracellular ratio between dUTP and dTTP, and contributes to genome stability. Nevertheless, the catalytic performance of Dr-dUTPase has not been reported. RESULTS: To confirm the catalytic activity of Dr-dUTPase, we cloned and expressed Dr-DUT in E. coli. Then, we purified Dr-dUTPase using His-tag and removed the tag with thrombin. The resulting Dr-dUTPase had the leading peptide Gly–Ser–His– originating from the vector at the amino terminus, and a mutation, Arg66Lys, to remove the internal thrombin site. We observed the hydrolysis of dUTP by Dr-dUTPase using Cresol Red as a proton sensor. The K(m) for dUTP was determined to be 4.0 µM, which is similar to that for human dUTPase. Dr-dUTPase exhibited a preference for dUTP over the other nucleotides. We conclude the Dr-dUTPase has catalytic activity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13104-019-4191-6) contains supplementary material, which is available to authorized users. BioMed Central 2019-03-22 /pmc/articles/PMC6431053/ /pubmed/30902068 http://dx.doi.org/10.1186/s13104-019-4191-6 Text en © The Author(s) 2019 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Note
Alam, Md. Shahanoor
Moriyama, Hideaki
Matsumoto, Midori
Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
title Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
title_full Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
title_fullStr Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
title_full_unstemmed Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
title_short Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
title_sort enzyme kinetics of dutpase from the planarian dugesia ryukyuensis
topic Research Note
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431053/
https://www.ncbi.nlm.nih.gov/pubmed/30902068
http://dx.doi.org/10.1186/s13104-019-4191-6
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