Müller Nadine, Khobta Andriy
Unit "Responses to DNA Lesions", Institute of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, 55131, Germany.
Unit "Responses to DNA Lesions", Institute of Toxicology, University Medical Center of the Johannes Gutenberg University Mainz, Mainz, 55131, Germany; Institute of Nutritional Science, University of Jena, Jena, 07743, Germany.
Redox Biol. 2021 Jul;43:101997. doi: 10.1016/j.redox.2021.101997. Epub 2021 Apr 30.
The oxidation-induced DNA modification 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was recently implicated in the activation and repression of gene transcription. We aimed at a systematic characterisation of the impacts of 8-oxodG on the activity of a GC box placed upstream from the RNA polymerase II core promoter. With the help of reporters carrying single synthetic 8-oxodG residues at four conserved G:C base pairs (underlined) within the 5'-TGGGCGGAGC-3' GC box sequence, we identified two modes of interference of 8-oxodG with the promoter activity. Firstly, 8-oxodG in the purine-rich (but not in the pyrimidine-rich) strand caused direct impairment of transcriptional activation. In addition, and independently of the first mechanism, 8-oxodG initiated a decline of the gene expression, which was mediated by the specific DNA glycosylase OGG1. For the different 8-oxodG positions, the magnitude of this effect reflected the excision preferences of OGG1. Thus, 8-oxodG seeded in the pyrimidine-rich strand was excised with the highest efficiency and caused the most pronounced decrease of the promoter activity. Conversely, 8-oxodG in the symmetric position within the same CpG dinucleotide, was poorly excised and induced no decline of the gene expression. Of note, abasic lesions caused gene silencing in both positions. By contrast, an uncleavable apurinic lesion in the pyrimidine-rich strand enhanced the GC box activity, suggesting that the AP endonuclease step provides a switch between the active versus repressed promoter states during base excision repair.
氧化诱导的DNA修饰8-氧代-7,8-二氢-2'-脱氧鸟苷（8-氧代脱氧鸟苷，8-oxodG）最近被认为与基因转录的激活和抑制有关。我们旨在系统地表征8-氧代脱氧鸟苷对位于RNA聚合酶II核心启动子上游的GC框活性的影响。借助在5'-TGGGCGGAGC-3' GC框序列内四个保守的G:C碱基对（下划线）处携带单个合成8-氧代脱氧鸟苷残基的报告基因，我们确定了8-氧代脱氧鸟苷干扰启动子活性的两种模式。首先，富含嘌呤（而非富含嘧啶）链中的8-氧代脱氧鸟苷直接损害转录激活。此外，与第一种机制无关，8-氧代脱氧鸟苷引发基因表达下降，这是由特定的DNA糖基化酶OGG1介导的。对于不同的8-氧代脱氧鸟苷位置，这种效应的大小反映了OGG1的切除偏好。因此，富含嘧啶链中植入的8-氧代脱氧鸟苷切除效率最高，导致启动子活性下降最为明显。相反，同一CpG二核苷酸对称位置的8-氧代脱氧鸟苷切除效果不佳，不会导致基因表达下降。值得注意的是，无碱基损伤在两个位置均导致基因沉默。相比之下，富含嘧啶链中不可切割的脱嘌呤损伤增强了GC框活性，这表明在碱基切除修复过程中，AP内切酶步骤在活性与抑制的启动子状态之间提供了一个转换开关。
