A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives
A semi-automated non-radioactive system for measuring recovery of RNA synthesis and unscheduled DNA synthesis using ethynyluracil derivatives
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DOI：
10.1016/j.dnarep.2010.01.015
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发表时间：
2010-05-04
期刊：
影响因子：
3.8
通讯作者：
Ogi, Tomoo
中科院分区：
文献类型：
--
作者：
Nakazawa, Yuka;Yamashita, Shunichi;Ogi, Tomoo
核苷酸切除修复（NER）可清除细胞DNA中的主要紫外线光损伤。在人类中，受损的NER活性是几种光敏性疾病的原因，其中之一是皮肤癌易感性疾病，着色性干皮病（XP）。测量NER活性的两种常用检测方法是“计划外DNA合成（UDS）”和“RNA合成恢复（RRS）”，后者是NER转录偶联修复子通路的特定测量方法。这两种检测方法都是研究肾内坏死以及诊断肾内坏死相关疾病的关键技术。直到最近，这些测定的可靠方法还包括测量放射性标记核苷的掺入。我们已经建立了非放射性的方法来测定UDS和RRS水平，结合了最近开发的炔偶联核苷类似物，5-乙基-2'-脱氧尿嘧啶（EdU）和5-乙基尿嘧啶（EU）。EdU和EU分别作为UDS中的h -3胸嘧啶和RRS中的h -3尿嘧啶的替代品。基于这些炔核苷和一个集成的图像分析仪，我们开发了一个半自动化的检测系统，用于ner活性。我们证明了该系统在淋巴母细胞样样本和原代成纤维细胞的ner活性评估中的实用性。还考虑了该系统在大规模sirna筛选新型NER缺陷以及常规XP诊断中的潜在应用。(C) 2010 Elsevier B.V.版权所有
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.