A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives.
Search this article
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.
DNA repair, 9(5), pp.506-516; 2010
Journal
-
- DNA repair
-
DNA repair 9 (5), 506-516, 2010-05-04
Elsevier B.V.
- Tweet
Keywords
Details 詳細情報について
-
- CRID
- 1050005822286256128
-
- NII Article ID
- 120006984626
-
- NII Book ID
- AA11032547
-
- ISSN
- 15687864
- 15687856
-
- HANDLE
- 10069/23067
-
- Text Lang
- en
-
- Article Type
- journal article
-
- Data Source
-
- IRDB
- CiNii Articles