Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis

Md Shahanoor Alam, Hideaki Moriyama, Midori Matsumoto

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

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.

Original languageEnglish
Article number163
JournalBMC Research Notes
Volume12
Issue number1
DOIs
Publication statusPublished - 2019 Mar 22

Keywords

  • Planarian
  • Regeneration
  • dUTPase

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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