Screening for novel factors involved in nucleotide excision repair

核苷酸切除修复（NER）从细胞DNA中去除主要的紫外光损伤。通常用于测量NER活性的两种测定法是“非程序DNA合成（UDS）”和"RNA合成恢复（RRS）“。这些测定的可靠方法涉及放射性标记核苷掺入的测量。我们已经建立了非放射性程序，通过掺入炔共轭核苷类似物、5-乙炔基-2 '-脱氧尿苷（EdU）和5-乙炔尿苷（EU）来测定UDS和RRS水平。我们通过这种非放射性测定系统评估了来自NER缺陷患者的几种成纤维细胞的UDS和RRS水平。我们的结果是在良好的协议与传统的检测方法，表明该系统提供了一个方便，但灵敏和准确的测定。我们还证明了这种技术的实用性，大规模的siRNA筛选，以确定新的NER因子。

Nucleotide excision repair (NER) removes the major UV-photolesions from cellular DNA. Two assays commonly used in measurement of NER activity are 'unscheduled DNA synthesis (UDS)', and 'recovery of RNA synthesis (RRS)'. Reliable methods for these assays involved measurements of incorporation of radio-labeled nucleosides. We have established non-radioactive procedures for determining UDS and RRS levels by incorporation of alkyne-conjugated nucleoside analogues, 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyuridine (EU). We assessed the UDS and RRS levels of several fibroblasts derived from NER-deficient patients by this non-radioactive assay system. Our results were in good agreement with those obtained by conventional assays, indicating that the system provides a convenient, but sensitive and accurate assay. We also demonstrated the utility of this technique for a large-scale siRNA-screening to identify novel NER factors.