Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA.
Turn-on DNA damage sensors for the direct detection of 8-oxoguanine and photoproducts in native DNA.
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DOI：
10.1021/ja1116606
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发表时间：
2011-08-17
影响因子：
15
通讯作者：
Ghosh, Indraneel
中科院分区：
文献类型：
--
作者：
Furman, Jennifer L.;Mok, Pui-Wing;Badran, Ahmed H.;Ghosh, Indraneel
所有生物体中遗传信息的完整性不断受到各种内源性和环境侮辱的威胁。为了应对这种风险，DNA损伤反应被用于修复损伤和保持基因组的完整性。然而，异常的DNA损伤反应可能会导致遗传不稳定和突变、致癌或细胞死亡。为了在天然DNA的背景下直接监测DNA损伤事件，我们设计了两个利用遗传片段的萤火虫荧光素酶(分裂荧光素酶)的新传感器。这些传感器由一个甲基-CpG结合结构域(MBD)连接到分裂的荧光素酶片段上，用于将传感器定位到DNA(基因组中50-80%的CpG二核苷酸位点在胞嘧啶上对称甲基化)，而损伤识别结构域连接到荧光素酶的互补片段上，以探测邻近的核苷酸是否存在损伤。具体地说，我们利用氧鸟嘌呤糖基酶1(OGG1)来检测由于暴露于活性氧物种而引起的8-氧鸟嘌呤，并利用受损DNA结合蛋白2(DDB2)来检测UVC光诱导的嘧啶二聚体的光产物。这两个传感器使用寡核苷酸、质粒和哺乳动物基因组DNA以及系统地暴露于各种环境侮辱的HeLa细胞进行了优化和验证，表明这种利用MBD指导的DNA定位的方法为快速分析与DNA损伤和修复相关的特定化学修饰提供了一种简单、敏感且潜在的通用方法。
The integrity of the genetic information in all living organisms is constantly threatened by a variety of endogenous and environmental insults. To counter this risk, the DNA-damage response is employed for repairing lesions and maintaining genomic integrity. However, an aberrant DNA-damage response can potentially lead to genetic instability and mutagenesis, carcinogenesis, or cell death. To directly monitor DNA damage events in the context of native DNA, we have designed two new sensors utilizing genetically fragmented firefly luciferase (split luciferase). The sensors are comprised of a methyl-CpG binding domain (MBD) attached to one fragment of split luciferase for localizing the sensor to DNA (50–80% of the CpG dinucleotide sites in the genome are symmetrically methylated at cytosines), while a damage-recognition domain is attached to the complementary fragment of luciferase to probe adjacent nucleotides for lesions. Specifically, we utilized oxoguanine glycosylase 1 (OGG1) to detect 8-oxoguanine caused by exposure to reactive oxygen species and employed the damaged-DNA binding protein 2 (DDB2) for detection of pyrimidine dimer photoproducts induced by UVC light. These two sensors were optimized and validated using oligonucleotides, plasmids, and mammalian genomic DNA, as well as HeLa cells that were systematically exposed to a variety of environmental insults, demonstrating that this methodology utilizing MBD-directed DNA localization provides a simple, sensitive, and potentially general approach for the rapid profiling of specific chemical modifications associated with DNA damage and repair.
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影响因子：
15
作者：
Jester, Benjamin W.;Cox, Kurt J.;Ghosh, Indraneel
通讯作者：
Ghosh, Indraneel
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