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

Nakazawa, Yuka, Yamashita, Shunichi, Lehmann, Alan R and Ogi, Tomoo (2010) A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives. DNA Repair, 9 (5). pp. 506-516. ISSN 1568-7864

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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 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.

Item Type: Article
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Depositing User: Alan Lehmann
Date Deposited: 06 Feb 2012 19:54
Last Modified: 30 Mar 2012 12:26
URI: http://sro.sussex.ac.uk/id/eprint/22927
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