期刊
DNA REPAIR
卷 9, 期 5, 页码 506-516出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.dnarep.2010.01.015
关键词
Nucleotide excision repair (NER); Transcription-coupled repair (TCR); Recovery of RNA synthesis (RRS); Unscheduled DNA synthesis (DOS); Xeroderma pigmentosum (XP); Cockayne syndrome (CS)
资金
- Yamada Apiculture Center Inc.
- ROHTO Pharmaceutical Co., Ltd.
- Japan Science and Technology Agency (JST)
- Japan Society for the Promotion of Science (JSPS) [20810021, 21810022]
- Ministry of Education, Culture, Sports, Sciences and Technology of Japan
- Medical Research Council [G0801130B] Funding Source: researchfish
- Grants-in-Aid for Scientific Research [22390189, 21810022, 20810021] Funding Source: KAKEN
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 H-3-thymidine in UDS and for H-3-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. (C) 2010 Elsevier B.V. All rights reserved.
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