A semi-automated non-radioactive 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 (3)H-thymidine in UDS and for (3)H-uridine in RRS. Based on these alkyne-nucleosides and an integrated image analyser, we have developed a semi-automated assay system for NER-activity. 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 large-scale siRNA-screening for novel NER defects as well as for routine XP diagnosis are also considered.
Nakazawa
Yuka
Y
Department of Molecular Medicine, Atomic Bomb Disease Institute, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan.
Yamashita
Shunichi
S
Lehmann
Alan R
AR
Ogi
Tomoo
T
D001331:Automation; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis*; D004260:DNA Repair; D005821:Genetic Techniques; D006801:Humans; D008856:Microscopy, Fluorescence; D012313:RNA / Q000096:biosynthesis*; D034741:RNA, Small Interfering / Q000235:genetics; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D014983:Xeroderma Pigmentosum / Q000473:pathology
D034741:RNA, Small Interfering; C073482:eniluracil; D014498:Uracil; D012313:RNA; D004247:DNA