Castillo-Acosta Víctor M, Estévez Antonio M, Vidal Antonio E, Ruiz-Perez Luis M, González-Pacanowska Dolores
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Avenida del Conocimiento, s/n 18100-Armilla, Granada, Spain.
Int J Biochem Cell Biol. 2008;40(12):2901-13. doi: 10.1016/j.biocel.2008.06.009. Epub 2008 Jul 5.
The enzyme deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is responsible for the control of intracellular levels of dUTP thus controlling the incorporation of uracil into DNA during replication. Trypanosomes and certain eubacteria contain a dimeric dUTP-dUDPase belonging to the recently described superfamily of all-alpha NTP pyrophosphatases which bears no resemblance with typical eukaryotic trimeric dUTPases and presents unique properties regarding substrate specificity and product inhibition. While the biological trimeric enzymes have been studied in detail and the human enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies, little is known regarding the biological function of dimeric proteins. Here, we show that in Trypanosoma brucei, the dimeric dUTPase is a nuclear enzyme and that down-regulation of activity by RNAi greatly reduces cell proliferation and increases the intracellular levels of dUTP. Defects in growth could be partially reverted by the addition of exogenous thymidine. dUTPase-depleted cells presented hypersensitivity to methotrexate, a drug that increases the intracellular pools of dUTP, and enhanced uracil-DNA glycosylase activity, the first step in base excision repair. The knockdown of activity produces numerous DNA strand breaks and defects in both S and G2/M progression. Multiple parasites with a single enlarged nucleus were visualized together with an enhanced population of anucleated cells. We conclude that dimeric dUTPases are strongly involved in the control of dUTP incorporation and that adequate levels of enzyme are indispensable for efficient cell cycle progression and DNA replication.
脱氧尿苷5'-三磷酸核苷酸水解酶（dUTPase）负责控制细胞内dUTP的水平，从而在复制过程中控制尿嘧啶掺入DNA。锥虫和某些真细菌含有一种二聚体dUTP-dUDPase，它属于最近描述的全α NTP焦磷酸酶超家族，与典型的真核三聚体dUTPase没有相似之处，并且在底物特异性和产物抑制方面具有独特的性质。虽然已经对生物三聚体酶进行了详细研究，并且有人提出人类酶是抗癌化疗策略中一个有前景的新靶点，但对于二聚体蛋白的生物学功能知之甚少。在这里，我们表明在布氏锥虫中，二聚体dUTPase是一种核酶，通过RNA干扰下调其活性会大大降低细胞增殖并增加细胞内dUTP的水平。添加外源性胸苷可以部分恢复生长缺陷。dUTPase缺失的细胞对甲氨蝶呤高度敏感，甲氨蝶呤是一种增加细胞内dUTP池的药物，并且增强了尿嘧啶-DNA糖基化酶的活性，这是碱基切除修复的第一步。活性的敲低会产生大量DNA链断裂以及S期和G2/M期进程的缺陷。可以看到多个具有单个增大细胞核的寄生虫以及无核细胞群体的增加。我们得出结论，二聚体dUTPase强烈参与dUTP掺入的控制，并且足够水平的酶对于有效的细胞周期进程和DNA复制是不可或缺的。
