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

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posted on 2023-06-07, 15:17 authored by Yuka Nakazawa, Shunichi Yamashita, Alan LehmannAlan Lehmann, Tomoo Ogi
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 3H-thymidine in UDS and for 3H-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.

History

Publication status

  • Published

File Version

  • Accepted version

Journal

DNA Repair

ISSN

1568-7864

Publisher

Elsevier

Issue

5

Volume

9

Page range

506-516

Department affiliated with

  • Sussex Centre for Genome Damage Stability Publications

Research groups affiliated with

  • Genome Damage and Stability Centre Publications

Notes

GDSC310

Full text available

  • Yes

Peer reviewed?

  • Yes

Legacy Posted Date

2010-04-08

First Open Access (FOA) Date

2018-03-16

First Compliant Deposit (FCD) Date

2019-07-02

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