Shafirovich Vladimir, Kropachev Konstantin, Anderson Thomas, Liu Zhi, Kolbanovskiy Marina, Martin Brooke D, Sugden Kent, Shim Yoonjung, Chen Xuejing, Min Jung-Hyun, Geacintov Nicholas E
From the Department of Chemistry, New York University, New York, New York 10003,
From the Department of Chemistry, New York University, New York, New York 10003.
J Biol Chem. 2016 Mar 4;291(10):5309-19. doi: 10.1074/jbc.M115.693218. Epub 2016 Jan 5.
The well known biomarker of oxidative stress, 8-oxo-7,8-dihydroguanine, is more susceptible to further oxidation than the parent guanine base and can be oxidatively transformed to the genotoxic spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) lesions. Incubation of 135-mer duplexes with single Sp or Gh lesions in human cell extracts yields a characteristic nucleotide excision repair (NER)-induced ladder of short dual incision oligonucleotide fragments in addition to base excision repair (BER) incision products. The ladders were not observed when NER was inhibited either by mouse monoclonal antibody (5F12) to human XPA or in XPC(-/-) fibroblast cell extracts. However, normal NER activity appeared when the XPC(-/-) cell extracts were complemented with XPC-RAD23B proteins. The Sp and Gh lesions are excellent substrates of both BER and NER. In contrast, 5-guanidino-4-nitroimidazole, a product of the oxidation of guanine in DNA by peroxynitrite, is an excellent substrate of BER only. In the case of mouse embryonic fibroblasts, BER of the Sp lesion is strongly reduced in NEIL1(-/-) relative to NEIL1(+/+) extracts. In summary, in human cell extracts, BER and NER activities co-exist and excise Gh and Sp DNA lesions, suggesting that the relative NER/BER product ratios may depend on competitive BER and NER protein binding to these lesions.
氧化应激的著名生物标志物8-氧代-7,8-二氢鸟嘌呤比母体鸟嘌呤碱基更容易进一步氧化，并可被氧化转化为具有基因毒性的螺环亚氨基二氢尿嘧啶（Sp）和5-胍基尿嘧啶（Gh）损伤。在人细胞提取物中，将含有单个Sp或Gh损伤的135聚体双链体进行孵育，除了碱基切除修复（BER）切口产物外，还会产生由核苷酸切除修复（NER）诱导的特征性短双切口寡核苷酸片段梯状条带。当用抗人XPA的小鼠单克隆抗体（5F12）或在XPC（-/-）成纤维细胞提取物中抑制NER时，未观察到这些梯状条带。然而，当用XPC-RAD23B蛋白补充XPC（-/-）细胞提取物时，会出现正常的NER活性。Sp和Gh损伤都是BER和NER的优良底物。相比之下，5-胍基-4-硝基咪唑是DNA中鸟嘌呤被过氧亚硝酸盐氧化的产物，仅是BER的优良底物。在小鼠胚胎成纤维细胞中，相对于NEIL1（+/+）提取物，NEIL1（-/-）中Sp损伤的BER活性大幅降低。总之，在人细胞提取物中，BER和NER活性共存并切除Gh和Sp DNA损伤，这表明NER/BER产物的相对比例可能取决于BER和NER蛋白与这些损伤的竞争性结合。
