Screening for novel factors involved in nucleotide excision repair

筛选参与核苷酸切除修复的新因子

• 批准号: 21810022
• 负责人: NAKAZAWA Yuka
• 金额: $ 1.7万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Research Activity Start-up
• 财政年份: 2009
• 资助国家: 日本
• 起止时间: 2009 至 2010
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-21810022/
• 关键词: DNA修復; ヌクレオチド除去修復; 紫外線; エチニルデオキシウリジン; エチニルウリジン; In Cell Analyzer

Nucleotide excision repair (NER) removes the major UV-photolesions from cellular DNA. Two assays commonly used in measurement of NER activity are 'unscheduled DNA synthesis (UDS)', and 'recovery of RNA synthesis (RRS)'. 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 alkyne-conjugated nucleoside analogues, 5-ethynyl-2'-deoxyuridine (EdU) and 5-ethynyuridine (EU). We assessed the UDS and RRS levels of several fibroblasts derived from NER-deficient patients by this non-radioactive assay system. Our results were in good agreement with those obtained by conventional assays, indicating that the system provides a convenient, but sensitive and accurate assay. We also demonstrated the utility of this technique for a large-scale siRNA-screening to identify novel NER factors.

核苷酸切除修复（NER）可清除细胞DNA中的主要紫外线光损伤。通常用于测量NER活性的两种检测方法是“计划外DNA合成（UDS）”和“RNA合成恢复（RRS）”。这些测定的可靠方法包括放射性标记核苷掺入的测量。我们建立了非放射性的方法，通过结合炔偶联核苷类似物，5-乙基-2'-脱氧尿嘧啶（EdU）和5-乙基尿嘧啶（EU）来测定UDS和RRS水平。我们通过这种非放射性检测系统评估了几种源自ner缺陷患者的成纤维细胞的UDS和RRS水平。结果与常规方法吻合较好，表明该方法简便、灵敏、准确。我们还展示了该技术在大规模sirna筛选中识别新型NER因子的实用性。

项目成果

EdUを用いたUDS測定法とXP診断への応用

使用 EdU 的 UDS 测量方法及其在 XP 诊断中的应用

• 发表时间: 2009
• 作者: 中沢由華.,S.Yamashita;A.Lehmann;T.Ogi.;Yasunori Sasakura・Miho M.Suzuki・Akiko Hozumi・Kazuo Inaba・Nori Satoh;白石陽子;松谷基和;山下真一;Yasunori Sasakura・Miho M.Suzuki・Akiko Hozumi・Kazuo Inaba・Nori Satoh;松永光平;白石陽子;中沢由華;松谷基和;山下真一;中沢由華;松谷基和;鈴木美穂;山下真一;白石陽子;中沢由華;鈴木美穂;山下真一;白石陽子;中沢由華;鈴木美穂;中沢由華;山下真一;白石陽子;鈴木美穂;中沢由華
• 通讯作者: 中沢由華

A rarid non-radioactive technique for measurement of repair synthesis in primary human fibroblasts by incorporation of ethynyl deoxyuridine(EdU)

一种通过掺入乙炔脱氧尿苷 (EdU) 测量原代人成纤维细胞修复合成的稀有非放射性技术

• 发表时间: 2009
• 作者: 中沢由華.,S.Yamashita;A.Lehmann;T.Ogi.;Yasunori Sasakura・Miho M.Suzuki・Akiko Hozumi・Kazuo Inaba・Nori Satoh;白石陽子;松谷基和;山下真一;Yasunori Sasakura・Miho M.Suzuki・Akiko Hozumi・Kazuo Inaba・Nori Satoh;松永光平;白石陽子;中沢由華;松谷基和;山下真一;中沢由華;松谷基和;鈴木美穂;山下真一;白石陽子;中沢由華;鈴木美穂;山下真一;白石陽子;中沢由華;鈴木美穂;中沢由華
• 通讯作者: 中沢由華

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

• DOI: 10.1016/j.dnarep.2010.01.015
• 发表时间: 2010-05-04
• 期刊: DNA REPAIR
• 影响因子: 3.8
• 作者: Nakazawa, Yuka;Yamashita, Shunichi;Ogi, Tomoo
• 通讯作者: Ogi, Tomoo

A rapid non-radioactive technique for measurement of nucleotide excision repair (NER) activity in primary human fibroblasts by incorporation of ethynyluracil derivatives

一种通过掺入乙炔尿嘧啶衍生物来测量原代人成纤维细胞中核苷酸切除修复 (NER) 活性的快速非放射性技术

• 发表时间: 2010
• 作者: 中沢由華.,S.Yamashita;A.Lehmann;T.Ogi.;Yasunori Sasakura・Miho M.Suzuki・Akiko Hozumi・Kazuo Inaba・Nori Satoh;白石陽子;松谷基和;山下真一;Yasunori Sasakura・Miho M.Suzuki・Akiko Hozumi・Kazuo Inaba・Nori Satoh;松永光平;白石陽子;中沢由華;松谷基和;山下真一;中沢由華
• 通讯作者: 中沢由華