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泄漏。两种常用的测定NER活性的方法是“非程序DNA合成(UDS)”和“RNA合成恢复(RRS)”。这些分析的可靠方法包括测量放射性标记的核苷的掺入。我们已经建立了非放射性的测定UDS和RRS水平的方法，通过掺入炔基连接的核苷类似物5-乙炔基-2‘-脱氧尿苷(EDU)和5-乙炔基尿苷(Eu)。我们用这个非放射性检测系统评估了来自NER缺乏患者的几个成纤维细胞的UDS和RRS水平。我们的结果与常规方法的结果一致，表明该系统提供了一种方便、灵敏和准确的分析方法。我们还展示了这项技术在大规模siRNA筛选中的应用，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.