Werner syndrome is a disorder characterized by a premature aging phenotype. The disease is caused by mutations in the WRN gene which encodes a DNA helicase/exonuclease which is involved in multiple aspects of DNA metabolism. Current methods mostly rely on radiometric techniques to assess WRN exonuclease activity. Here we present an alternative, quantitative approach based on non-radioactive isotope dilution mass spectrometry (LC-MS/MS). A oligoduplex substrate mimicking the telomeric sequence was used for method development. Released nucleotides, which correlate with the degree of oligoduplex degradation, were dephosphorylated, purified, and quantified by LC-MS/MS. Heavy-isotope-labeled internal standards were used to account for technical variability. The method was validated in terms of reproducibility, time-course and concentration-dependency of the reaction. As shown in this study, the LC-MS/MS method can assess exonuclease activity of WRN mutants, WRN's substrate and strand specificity, and modulatory effects of WRN interaction partners and posttranslational modifications. Moreover, it can be used to analyze the selectivity and processivity of WRN exonuclease and allows the screening of small molecules for WRN exonuclease inhibitors. Importantly, this approach can easily be adapted to study nucleases other than WRN. This is of general interest, because exonucleases are key players in DNA metabolism and aging mechanisms.

中文翻译：

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通过同位素稀释质谱法定量分析 WRN 核酸外切酶活性。

Werner 综合征是一种以过早衰老表型为特征的疾病。该疾病是由 WRN 基因突变引起的，该基因编码 DNA 解旋酶/外切核酸酶，该基因参与 DNA 代谢的多个方面。目前的方法主要依靠辐射测量技术来评估 WRN 核酸外切酶活性。在这里，我们提出了一种基于非放射性同位素稀释质谱 (LC-MS/MS) 的替代定量方法。模拟端粒序列的寡双链底物用于方法开发。释放的核苷酸与寡双链体降解程度相关，经过去磷酸化、纯化和 LC-MS/MS 定量。使用重同位素标记的内标来解释技术可变性。该方法在重现性方面得到验证，反应的时间过程和浓度依赖性。如本研究所示，LC-MS/MS 方法可以评估 WRN 突变体的核酸外切酶活性、WRN 的底物和链特异性，以及 WRN 相互作用伙伴和翻译后修饰的调节作用。此外，它可用于分析警告外切酶的选择性和持续合成能力，并允许筛选警告外切酶抑制剂的小分子。重要的是，这种方法可以很容易地适用于研究 WRN 以外的核酸酶。这是普遍感兴趣的，因为核酸外切酶是 DNA 代谢和衰老机制的关键参与者。WRN 相互作用伙伴和翻译后修饰的调节作用。此外，它可用于分析警告外切酶的选择性和持续合成能力，并允许筛选警告外切酶抑制剂的小分子。重要的是，这种方法可以很容易地适用于研究 WRN 以外的核酸酶。这是普遍感兴趣的，因为核酸外切酶是 DNA 代谢和衰老机制的关键参与者。WRN 相互作用伙伴和翻译后修饰的调节作用。此外，它可用于分析警告外切酶的选择性和持续合成能力，并允许筛选警告外切酶抑制剂的小分子。重要的是，这种方法可以很容易地适用于研究 WRN 以外的核酸酶。这是普遍感兴趣的，因为核酸外切酶是 DNA 代谢和衰老机制的关键参与者。