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DNA Repair (Amst). 2010 May 4;9(5):506-16. doi: 10.1016/j.dnarep.2010.01.015. Epub 2010 Feb 18.

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

Author information

1
Department of Molecular Medicine, Atomic Bomb Disease Institute, Graduate School of Biomedical Sciences, Nagasaki University, 1-12-4, Sakamoto, Nagasaki 852-8523, Japan.

Abstract

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.

PMID:
20171149
DOI:
10.1016/j.dnarep.2010.01.015
[Indexed for MEDLINE]

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