Repair of oxidative DNA damage in vitro:: A tool for screening antioxidative compounds
Repair of oxidative DNA damage in vitro:: A tool for screening antioxidative compounds
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DOI：
10.1016/s0278-6915(99)00094-0
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发表时间：
1999-09-01
影响因子：
4.3
通讯作者：
Calsou, P
中科院分区：
文献类型：
--
作者：
Salles, B;Sattler, U;Calsou, P
活性氧物种(ROS)引发碱基DNA改变的形成，这些改变是通过切除损伤的步骤进行处理的，然后是修复、合成和连接步骤，以恢复链的连续性。我们之前已经报道了用体外化学发光DNA修复合成试验(Salles等人)检测DNA加合物。1995)，它允许通过吸附在敏化微孔板上的受损的质粒DNA中的无细胞提取物来测量修复合成。3D(DNA损伤检测)检测是在生物素-dUTP存在的情况下进行的，生物素-dUTP在修复合成步骤中被掺入。用ExtrAvidin-辣根过氧化物酶酶联免疫吸附试验和化学发光法测定修复合成的程度。3D分析允许检测任何类型的碱基变化，包括碱基氧化。有趣的是，在ROS的受控生产下，可以用3D分析进行抗氧化剂的筛选程序。利用质粒DNA吸附的优势，通过与特异性抗体的酶联免疫吸附和化学发光法(4D法)检测到的大肠杆菌FpG蛋白可以识别氧化碱基损伤。结合抗氧化剂的筛选过程，讨论了抗氧化剂检测方法的发展及存在的问题。(C)1999爱思唯尔科学有限公司。保留所有权利。
Reactive oxygen species (ROS) provoke the formation of base DNA alterations that are processed by an excision step of the lesion followed by a repair synthesis and ligation step to restore the strand continuity. We have reported previously the detection of DNA adducts by an in vitro chemiluminescence DNA repair synthesis assay (Salles er al.. 1995) which allows the measurement of repair synthesis by cell-free extracts in damaged plasmid DNA adsorbed on sensitized microplate wells. The 3D (DNA damage detection) assay was performed in the presence of biotin-dUTP which was incorporated during the repair synthesis step. The extent of repair synthesis was measured in an ELISA reaction with ExtrAvidin-horse radish peroxidase and chemiluminescence detection. The 3D assay allows detection of any type of base alterations including base oxidation. Interestingly, under controlled production of ROS a screening procedure of antioxidants might be carried out with the 3D assay. By taking advantage of plasmid DNA adsorption, oxidative base damage can be recognized by the Escherichia coli Fpg protein which was detected in an ELISA reaction with specific antibody and chemiluminescence measurement (4D assay). With the sceening procedure of antioxidative compounds in mind, the development of such assays and their drawbacks are discussed. (C) 1999 Elsevier Science Ltd. All rights, reserved.