Institute of Enzymology, RCNS, Hungarian Academy of Sciences, H-1117 Budapest, Hungary.
Doctoral School of Multidisciplinary Medical Science, University of Szeged, H-6720 Szeged, Hungary.
Biomolecules. 2019 Apr 4;9(4):136. doi: 10.3390/biom9040136.
Sanitization of nucleotide pools is essential for genome maintenance. Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is a key enzyme in this pathway since it catalyzes the cleavage of 2'-deoxyuridine 5'-triphosphate (dUTP) into 2'-deoxyuridine 5'-monophosphate (dUMP) and inorganic pyrophosphate. Through its action dUTPase efficiently prevents uracil misincorporation into DNA and at the same time provides dUMP, the substrate for de novo thymidylate biosynthesis. Despite its physiological significance, knock-out models of dUTPase have not yet been investigated in mammals, but only in unicellular organisms, such as bacteria and yeast. Here we generate CRISPR/Cas9-mediated dUTPase knock-out in mice. We find that heterozygous +/- animals are viable while having decreased dUTPase levels. Importantly, we show that dUTPase is essential for embryonic development since early -/- embryos reach the blastocyst stage, however, they die shortly after implantation. Analysis of pre-implantation embryos indicates perturbed growth of both inner cell mass (ICM) and trophectoderm (TE). We conclude that dUTPase is indispensable for post-implantation development in mice.
核苷酸池的净化对于基因组的维护至关重要。脱氧尿苷 5'-三磷酸核苷水解酶（dUTPase）是该途径中的关键酶，因为它催化 2'-脱氧尿苷 5'-三磷酸（dUTP）裂解为 2'-脱氧尿苷 5'-单磷酸（dUMP）和焦磷酸。通过其作用，dUTPase 有效地防止尿嘧啶错误掺入 DNA，同时提供 dUMP，这是从头胸苷酸生物合成的底物。尽管其具有生理意义，但 dUTPase 的敲除模型尚未在哺乳动物中进行研究，而仅在单细胞生物中，如细菌和酵母中进行了研究。在这里，我们通过 CRISPR/Cas9 介导的方法在小鼠中产生了 dUTPase 敲除。我们发现杂合子 +/-动物是存活的，尽管它们的 dUTPase 水平降低了。重要的是，我们表明 dUTPase 对于胚胎发育是必不可少的，因为早期 -/- 胚胎达到囊胚阶段，但在植入后不久就死亡。对植入前胚胎的分析表明，内细胞团（ICM）和滋养外胚层（TE）的生长都受到了干扰。我们得出结论，dUTPase 对于小鼠的植入后发育是不可或缺的。
