Sarmini Leen, Kitsera Nataliya, Meabed Mohammed, Khobta Andriy
Institute of Nutritional Sciences, Friedrich Schiller University Jena, Jena, Germany.
Institute of Toxicology, University Medical Center Mainz, Mainz, Germany.
J Biol Chem. 2025 Mar 27;301(5):108459. doi: 10.1016/j.jbc.2025.108459.
The N position of guanine is a preferential reaction site in DNA for numerous dietary and environmental carcinogens or their electrophilic metabolites, aldehydes arising from lipid peroxidation as well as reactive by-products of normal metabolism. However, DNA repair mechanisms of the resulting covalent adducts in mammalian cells are not well understood, with nucleotide excision repair (NER), base excision repair, and a dioxygenase-mediated damage reversal being discussed as likely pathways. Considering fundamentally different damage recognition principles between the global genome NER and the transcription-coupled (TC)-NER, we here assessed transcription blocking capacities of four synthetic deoxyguanosine (dGuo) adducts of variable size and geometry, using a transfection-based reporter assay. Notably, adducts as different as the aliphatic N-ethylguanine, the exocyclic 1,N-ethenoguanine, and the bulky polycyclic 3-(deoxyguanosin-N-yl)-2-acetylaminofluorene, displayed robust DNA strand-specific transcription-blocking properties. The specific TC-NER components ERCC8/CSA and ERCC6/CSB were consistently required for the removal of all transcription-blocking N-dGuo adducts, whereas the absence of XPC or DDB2/XPE (both specific to global genome NER) did not compromise the repair capacities in the isogenic human cell models. In contrast, no inhibition of the gene expression was detected for reporter constructs carrying N-methylguanine even in the NER-deficient XP-A cell line, suggesting that this adduct is either bypassed with very high efficiency during transcription or repaired by a mechanism different from NER. Collectively, the results identify N-dGuo adducts bigger than methylguanine as a structural subclass of transcription-blocking DNA lesions whose repair heavily relies on the TC-NER pathway.
鸟嘌呤的N位是DNA中众多膳食和环境致癌物或其亲电代谢产物、脂质过氧化产生的醛以及正常代谢的反应性副产物的优先反应位点。然而，哺乳动物细胞中由此产生的共价加合物的DNA修复机制尚不完全清楚，核苷酸切除修复（NER）、碱基切除修复和双加氧酶介导的损伤逆转被认为是可能的途径。考虑到全基因组NER和转录偶联（TC）-NER之间根本不同的损伤识别原则，我们在这里使用基于转染的报告基因检测方法评估了四种大小和几何形状不同的合成脱氧鸟苷（dGuo）加合物的转录阻断能力。值得注意的是，诸如脂肪族N-乙基鸟嘌呤、外环1,N-乙烯基鸟嘌呤和庞大的多环3-（脱氧鸟苷-N-基）-2-乙酰氨基芴等不同的加合物都表现出强大的DNA链特异性转录阻断特性。去除所有转录阻断性N-dGuo加合物始终需要特定的TC-NER组分ERCC8/CSA和ERCC6/CSB，而XPC或DDB2/XPE（两者均为全基因组NER所特有的）的缺失在同基因人类细胞模型中并不影响修复能力。相比之下，即使在NER缺陷的XP-A细胞系中，携带N-甲基鸟嘌呤的报告基因构建体也未检测到基因表达受到抑制，这表明该加合物在转录过程中要么以非常高的效率被绕过，要么通过不同于NER的机制进行修复。总的来说，这些结果确定大于甲基鸟嘌呤的N-dGuo加合物是一类转录阻断性DNA损伤的结构亚类，其修复在很大程度上依赖于TC-NER途径。
