School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
DNA Repair (Amst). 2010 Feb 4;9(2):144-52. doi: 10.1016/j.dnarep.2009.11.009. Epub 2009 Dec 29.
An abundant form of DNA damage caused by reactive oxygen species is 8-oxo-7,8-dihydroguanine for which the base excision repair protein 8-oxoguanine-DNA glycosylase 1 (OGG1) is a major repair enzyme. To assess the location and intracellular activity of the OGG1 protein in response to oxidative stress, we have utilised a fluorescence-quench molecular beacon switch containing a 8-oxo-dG:C base pair and a fluorescent and quencher molecule at opposite ends of a hairpin oligonucleotide. Oxidative stress was induced by treatment with potassium bromate. Flow cytometry demonstrated a concentration-dependent increase in the activity of OGG1 that was detected by the fluorescence produced when the oligonucleotide was cleaved in the cells treated with potassium bromate. This signal is highly specific and not detectable in OGG1 knock out cells. Induction of OGG1 activity is not a result of induction of OGG1 gene expression as assessed by qPCR suggesting a role for protein stabilisation or increased OGG1 catalytic activity. High resolution confocal microscopy pinpointed the location of the fluorescent molecular beacon in live cells to perinuclear regions that were identified as mitochondria by co-staining with mitotracker dye. There is no evidence of cut beacon within the nuclear compartment of the cell. Control experiments with a positive control beacon (G:C base pair and lacking the DAB quencher) did not result in mitochondrial localisation of fluorescence signal indicating that the dye does not accumulate in mitochondria independent of OGG1 activity. Furthermore, faint nuclear staining was apparent confirming that the beacon structure is able to enter the nucleus. In conclusion, these data indicate that the mitochondria are the major site for OGG1 repair activity under conditions of oxidative stress.
活性氧引起的大量 DNA 损伤形式是 8-氧代-7,8-二氢鸟嘌呤，碱基切除修复蛋白 8-氧代鸟嘌呤-DNA 糖基化酶 1（OGG1）是主要的修复酶。为了评估 OGG1 蛋白在应对氧化应激时的位置和细胞内活性，我们利用了一种荧光猝灭分子信标开关，其中包含一个 8-氧代-dG:C 碱基对和一个荧光和猝灭分子在发夹寡核苷酸的两端。用溴酸钾处理诱导氧化应激。流式细胞术显示 OGG1 活性呈浓度依赖性增加，在用溴酸钾处理的细胞中，当寡核苷酸被切割时，会产生荧光来检测这种活性。这种信号具有高度特异性，在 OGG1 敲除细胞中无法检测到。如 qPCR 评估所示，OGG1 活性的诱导不是 OGG1 基因表达诱导的结果，这表明可能是蛋白质稳定或增加 OGG1 催化活性的结果。高分辨率共聚焦显微镜将荧光分子信标的位置精确定位在活细胞中的核周区域，这些区域通过与线粒体染料共染色被鉴定为线粒体。在细胞的核区没有切割信标的证据。用阳性对照信标（含有 G:C 碱基对且缺少 DAB 猝灭剂）进行的对照实验不会导致荧光信号在线粒体中的定位，这表明染料不会独立于 OGG1 活性在线粒体中积累。此外，明显的核染色较弱，证实了信标结构能够进入细胞核。总之，这些数据表明，在线粒体中是 OGG1 修复活性的主要部位在氧化应激条件下。
