Fluorescent probes for the analysis of DNA strand scission in base excision repair.
Fluorescent probes for the analysis of DNA strand scission in base excision repair.
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DOI：
10.1093/nar/gkq022
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发表时间：
2010-04
影响因子：
14.9
通讯作者：
Iwai S
中科院分区：
文献类型：
--
作者：
Matsumoto N;Toga T;Hayashi R;Sugasawa K;Katayanagi K;Ide H;Kuraoka I;Iwai S
我们开发了荧光探针，用于检测氧化损伤碱基切除修复中的链断裂。它们是发夹形寡核苷酸，每个都含有胸腺嘧啶乙二醇或5,6-二氢胸腺嘧啶的异构体作为中心的受损碱基，分别在5'-和3'-末端具有荧光团和猝灭剂。当损伤位点处的磷酸二酯键被酶裂解时，检测到荧光，因为带有荧光团的短片段在孵育温度下不能保持与分子其余部分杂交的双链体形式。使用这些探针测定的大肠杆菌核酸内切酶 III 及其人类同源物 NTH1 的底物特异性与之前使用 32P 标记底物通过凝胶电泳测定的底物特异性一致。动力学参数也已通过该方法测定。由于不同的荧光团附着在包含每个病变的寡核苷酸上，因此通过改变激发和检测波长，在单管中分别分析两种类型底物的反应。这些探针在与细胞提取物一起孵育的过程中被降解。因此，掺入硫代磷酸酯键以保护探针免受非特异性核酸酶的影响，并在 HeLa 细胞中成功检测到碱基切除修复活性。
We have developed fluorescent probes for the detection of strand scission in the excision repair of oxidatively damaged bases. They were hairpin-shaped oligonucleotides, each containing an isomer of thymine glycol or 5,6-dihydrothymine as a damaged base in the center, with a fluorophore and a quencher at the 5′- and 3′-ends, respectively. Fluorescence was detected when the phosphodiester linkage at the damage site was cleaved by the enzyme, because the short fragment bearing the fluorophore could not remain in a duplex form hybridized to the rest of the molecule at the incubation temperature. The substrate specificities of Escherichia coli endonuclease III and its human homolog, NTH1, determined by using these probes agreed with those determined previously by gel electrophoresis using 32P-labeled substrates. Kinetic parameters have also been determined by this method. Since different fluorophores were attached to the oligonucleotides containing each lesion, reactions with two types of substrates were analyzed separately in a single tube, by changing the excitation and detection wavelengths. These probes were degraded during an incubation with a cell extract. Therefore, phosphorothioate linkages were incorporated to protect the probes from nonspecific nucleases, and the base excision repair activity was successfully detected in HeLa cells.
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