Institut de Biologie des Plantes, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8618, Université Paris-Sud 11, Orsay, France.
PLoS One. 2011 Apr 26;6(4):e18658. doi: 10.1371/journal.pone.0018658.
Deoxyuridine triphosphatase (dUTPase) enzyme is an essential enzyme that protects DNA against uracil incorporation. No organism can tolerate the absence of this activity. In this article, we show that dUTPase function is conserved between E. coli (Escherichia coli), yeast (Saccharomyces cerevisiae) and Arabidopsis (Arabidopsis thaliana) and that it is essential in Arabidopsis as in both micro-organisms. Using a RNA interference strategy, plant lines were generated with a diminished dUTPase activity as compared to the wild-type. These plants are sensitive to 5-fluoro-uracil. As an indication of DNA damage, inactivation of dUTPase results in the induction of AtRAD51 and AtPARP2, which are involved in DNA repair. Nevertheless, RNAi/DUT1 constructs are compatible with a rad51 mutation. Using a TUNEL assay, DNA damage was observed in the RNAi/DUT1 plants. Finally, plants carrying a homologous recombination (HR) exclusive substrate transformed with the RNAi/DUT1 construct exhibit a seven times increase in homologous recombination events. Increased HR was only detected in the plants that were the most sensitive to 5-fluoro-uracils, thus establishing a link between uracil incorporation in the genomic DNA and HR. Our results show for the first time that genetic instability provoked by the presence of uracils in the DNA is poorly tolerated and that this base misincorporation globally stimulates HR in plants.
脱氧尿苷三磷酸酶（dUTPase）是一种保护 DNA 免受尿嘧啶掺入的必需酶。没有任何生物体可以容忍这种活性的缺失。在本文中，我们表明 dUTPase 功能在大肠杆菌（Escherichia coli）、酵母（Saccharomyces cerevisiae）和拟南芥（Arabidopsis thaliana）之间是保守的，并且它在拟南芥中与在两种微生物中一样是必需的。使用 RNA 干扰策略，与野生型相比，植物系产生了 dUTPase 活性降低的植物系。这些植物对 5-氟尿嘧啶敏感。作为 DNA 损伤的一个指标，dUTPase 的失活导致 AtRAD51 和 AtPARP2 的诱导，这两者都参与 DNA 修复。然而，RNAi/DUT1 构建体与 rad51 突变兼容。使用 TUNEL 测定法，在 RNAi/DUT1 植物中观察到 DNA 损伤。最后，携带同源重组（HR）专用底物的植物用 RNAi/DUT1 构建体转化，导致同源重组事件增加了七倍。仅在对 5-氟尿嘧啶最敏感的植物中检测到 HR 的增加，从而在 DNA 中尿嘧啶的掺入与 HR 之间建立了联系。我们的结果首次表明，DNA 中尿嘧啶的存在引起的遗传不稳定性很难被容忍，并且这种碱基错误掺入会全局刺激植物中的 HR。
