Chemistry Department , New York University , 31 Washington Place , New York , New York 10003-5180 , United States.
Chem Res Toxicol. 2019 Apr 15;32(4):753-761. doi: 10.1021/acs.chemrestox.8b00411. Epub 2019 Feb 8.
The interchange between different repair mechanisms in human cells has long been a subject of interest. Here, we provide a direct demonstration that the oxidatively generated guanine lesions spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) embedded in double-stranded DNA are substrates of both base excision repair (BER) and nucleotide excision repair (NER) mechanisms in intact human cells. Site-specifically modified, P-internally labeled double-stranded DNA substrates were transfected into fibroblasts or HeLa cells, and the BER and/or NER mono- and dual incision products were quantitatively recovered after 2-8 h incubation periods and lysis of the cells. DNA duplexes bearing single benzo[ a]pyrene-derived guanine adduct were employed as positive controls of NER. The NER activities, but not the BER activities, were abolished in XPA cells, while the BER yields were strongly reduced in NEIL1 cells. Co-transfecting different concentrations of analogous DNA sequences bearing the BER substrates 5-hydroxyuracil diminish the BER yields of Sp lesions and enhance the yields of NER products. These results are consistent with a model based on the local availability of BER and NER factors in human cells and their competitive binding to the same Sp or Gh BER/NER substrates.
不同修复机制在人类细胞中的相互作用一直是研究的热点。在这里，我们直接证明了氧化生成的鸟嘌呤损伤螺环亚氨基二氢嘧啶（Sp）和 5-胍基尿嘧啶（Gh）嵌入双链 DNA 是碱基切除修复（BER）和核苷酸切除修复（NER）机制的底物。特异性修饰的、P 位内部标记的双链 DNA 底物被转染到成纤维细胞或 HeLa 细胞中，在 2-8 小时孵育期后和细胞裂解，定量回收 BER 和/或 NER 单和双切口产物。含有单个苯并[ a]芘衍生鸟嘌呤加合物的 DNA 双链体被用作 NER 的阳性对照。在 XPA 细胞中，NER 活性而不是 BER 活性被消除，而在 NEIL1 细胞中 BER 产率则大大降低。共转染不同浓度的类似含有 BER 底物 5-羟脲嘧啶的 DNA 序列，会降低 Sp 损伤的 BER 产率，并提高 NER 产物的产率。这些结果与基于人类细胞中 BER 和 NER 因子的局部可用性及其对相同 Sp 或 Gh BER/NER 底物的竞争结合的模型一致。
