Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Parque Tecnológico de Ciencias de la Salud, Armilla (Granada), Spain.
Int J Biochem Cell Biol. 2012 Sep;44(9):1555-68. doi: 10.1016/j.biocel.2012.06.014. Epub 2012 Jun 21.
Cells contain low amounts of uracil in DNA which can be the result of dUTP misincorporation during replication or cytosine deamination. Elimination of uracil in the base excision repair pathway yields an abasic site, which is potentially mutagenic unless repaired. The Trypanosoma brucei genome presents a single uracil-DNA glycosylase responsible for removal of uracil from DNA. Here we establish that no excision activity is detected on U:G, U:A pairs or single-strand uracil-containing DNA in uracil-DNA glycosylase null mutant cell extracts, indicating the absence of back-up uracil excision activities. While procyclic forms can survive with moderate amounts of uracil in DNA, an analysis of the mutation rate and spectra in mutant cells revealed a hypermutator phenotype where the predominant events were GC to AT transitions and insertions. Defective elimination of uracil via the base excision repair pathway gives rise to hypersensitivity to antifolates and oxidative stress and an increased number of DNA strand breaks, suggesting the activation of alternative DNA repair pathways. Finally, we show that uracil-DNA glycosylase defective cells exhibit reduced infectivity in vivo demonstrating that efficient uracil elimination is important for survival within the mammalian host.
细胞中的 DNA 中含有少量的尿嘧啶，这可能是复制过程中 dUTP 错误掺入或胞嘧啶脱氨的结果。碱基切除修复途径中尿嘧啶的消除会产生无碱基位点，如果不修复，就有可能产生突变。布氏锥虫基因组中存在一种单尿嘧啶-DNA 糖基化酶，负责从 DNA 中去除尿嘧啶。在这里，我们确定在尿嘧啶-DNA 糖基化酶缺失突变体细胞提取物中，未检测到 U:G、U:A 对或单链含尿嘧啶的 DNA 的切除活性，这表明不存在备用的尿嘧啶切除活性。虽然前鞭毛体可以在 DNA 中有适量尿嘧啶的情况下存活，但对突变细胞中突变率和谱的分析显示出一种超突变表型，其中主要事件是 GC 向 AT 的转换和插入。碱基切除修复途径中尿嘧啶的消除缺陷导致对叶酸类似物和氧化应激的敏感性增加以及 DNA 链断裂数量增加，这表明激活了替代的 DNA 修复途径。最后，我们表明，尿嘧啶-DNA 糖基化酶缺陷细胞在体内的感染性降低，表明有效的尿嘧啶消除对于在哺乳动物宿主中存活很重要。
