Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
Enzyme kinetics of dUTPase from the planarian Dugesia ryukyuensis
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DOI：
10.1186/s13104-019-4191-6
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发表时间：
2019-03-22
影响因子：
1.8
通讯作者：
Matsumoto, Midori
中科院分区：
文献类型：
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作者：
Alam, Md. Shahanoor;Moriyama, Hideaki;Matsumoto, Midori
目的：以琉球三角涡虫（Dugesia ryukyuensis，Dr）为代表的真涡虫具有很强的再生能力，这需要DNA复制的增强。敲除编码脱氧尿苷5 '-三磷酸焦磷酸酶（dUTR）的Dr中的DUT基因，促进DNA片段化，抑制再生，并最终导致死亡。dUTP酶催化dUTP水解为dUMP和焦磷酸盐。已知dUTR通过平衡dUTP和dTTP之间的细胞内比率来防止尿嘧啶错误掺入DNA中，并且有助于基因组稳定性。结果：为了证实Dr-dUTR的催化活性，我们克隆了Dr-dUTR，并在E.杆菌然后，我们使用His-标签纯化Dr-dUTR，并用凝血酶去除标签。所得到的Dr-dUTR在氨基末端具有源自载体的前导肽Gly-Ser-His-，和突变Arg 66 Lys，以去除内部凝血酶位点。以甲酚红为质子传感器，观察了Dr-dUTR对dUTP的水解。测定dUTP的Km为4.0 μ M，与人dUTP相似。Dr-dUTR显示出对dUTP的偏好超过其他核苷酸。我们认为Dr-dUTR具有催化活性。
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 mu M, 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.