Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling
Detection of DNA damage in prokaryotes by terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling
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DOI：
10.1128/aem.66.3.1001-1006.2000
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发表时间：
2000-03-01
影响因子：
4.4
通讯作者：
Azam, F
中科院分区：
文献类型：
--
作者：
Rohwer, F;Azam, F
许多试剂可以损伤环境中原核生物的DNA（例如，活性氧、辐射和次级代谢物如抗生素、酶、饥饿等）。大量潜在的DNA损伤剂，以及它们的不同作用模式，排除了基于核酸分解产物检测的DNA损伤的简单测试。在这项研究中，游离的3 '-OH DNA末端，由直接损伤或切除DNA修复产生，用于评估DNA损伤。末端脱氧核糖核苷酸转移酶（TdT）介导的dUTP缺口末端标记（TUNEL）是一种用异硫氰酸荧光素（dUTP-fluorescein isothiocyanate）酶标记DNA末端的方法，用荧光显微镜或流式细胞术检测标记细胞，用TUNEL法检测过氧化氢对古菌Haloferax volcano ii和大肠杆菌Escherichia coli的DNA损伤，蛋白质合成抑制剂氯霉素和白喉毒素阻断了大肠杆菌和H. volcanii，分别。用TUNEL法检测紫外线和噬菌体感染引起的DNA损伤。这种方法在DNA损伤和修复的应用中应该是有用的，包括突变体筛选和监测环境中的DNA损伤。
Numerous agents can damage the DNA of prokaryotes in the environment (e.g., reactive oxygen species, irradiation, and secondary metabolites such as antibiotics, enzymes, starvation, etc.). The large number of potential DNA-damaging agents, as well as their diverse modes of action, precludes a simple test of DNA damage based on detection of nucleic acid breakdown products. In this study, free 3'-OH DNA ends, produced by either direct damage or excision DNA repair, were used to assess DNA damage. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) is a procedure in which 3'-OH DNA ends are enzymatically labeled with dUTP-fluorescein isothiocyanate using TdT, Cells labeled by this method can be detected using fluorescence microscopy or flow cytometry, TUNEL was used to measure hydrogen peroxide-induced DNA damage in the archaeon Haloferax volcanii and the bacterium Escherichia coli, DNA repair systems were implicated in the hydrogen peroxide-dependent generation of 3'-OH DNA ends by the finding that the protein synthesis inhibitors chloramphenicol and diphtheria toxin blocked TUNEL labeling of E, coli and H. volcanii, respectively. DNA damage induced by UV light and bacteriophage infection was also measured using TUNEL. This methodology should be useful in applications where DNA damage and repair are of interest, including mutant screening and monitoring of DNA damage in the environment.