Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis

Abstract

ObjectivePlanarians 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.ResultsTo 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 Km 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 materialThe online version of this article (10.1186/s13104-019-4191-6) contains supplementary material, which is available to authorized users.

Recent advances and application of whole genome amplification in molecular diagnosis and medicine

“…Presently, the mixed enzymes used in LA‐PCR include thermostable deoxyuridine 5'‐triphosphate pyrophosphatase (dUTPase) in addition to the above two enzymes, one enzyme does not contain 3'‐5' exonuclease activity and the other enzyme contains 3'‐5' exonuclease activity. 36 The deamination of deoxycytidine triphosphate (dCTP) will occur in the process of PCR and deoxyurodine triphosphate (dUTP). DNA polymerase with correction function irreversibly binds to deoxyuracil in vitro, which affects the efficiency of the enzyme and leads to the failure of LA‐PCR amplification.…”

“…Based on this principle, a high level of thermostable DNA polymerase without exonuclease activity and a small amount of DNA polymerase with exonuclease activity were mixed, and the extension ability of the former and the correction ability of the latter were used to successfully amplify the long fragments of the two kinds of enzymes under appropriate proportions and reaction conditions. Presently, the mixed enzymes used in LA‐PCR include thermostable deoxyuridine 5'‐triphosphate pyrophosphatase (dUTPase) in addition to the above two enzymes, one enzyme does not contain 3'‐5' exonuclease activity and the other enzyme contains 3'‐5' exonuclease activity 36 . The deamination of deoxycytidine triphosphate (dCTP) will occur in the process of PCR and deoxyurodine triphosphate (dUTP).…”