A semi-automated non-radiactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives

Nucleotide excision repair (NER) removes the major UV photolesions from cellular DNA. In humans, compromised NER activity is the cause of several photosensitive diseases, one of which is the skin-cancer predisposition disorder, xeroderma pigmentosum (XP). Two assays commonly used in measurement of NER activity are 'Unscheduled DNA synthesis (UDS)', and 'Recovery of RNA synthesis (RRS)', the latter being a specific measure of the transcription-coupled repair sub-pathway of NER. Both assays are key techniques for research in NER as well as in diagnoses of NER-related disorders. Until very recently, 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 recently-developed alkyne-conjugated nucleoside analogues, 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyuridine (EU). EdU and EU are respectively used as alternatives for Hthymidine in UDS and for H-uridine in RRS. Based on these alkyne-nucleosides and an integrated image analyser, we have developed a semi-automated assay system for NERactivity. We demonstrate the utility of this system for NER-activity assessments of lymphoblastoid samples as well as primary fibroblasts. Potential use of the system for largescale siRNA screening for novel NER defects as well as for routine XP diagnosis are also considered.