Screening for novel factors involved in nucleotide excision repair

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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.