Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts.
Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts.
复制标题
DOI：
10.1093/nar/29.8.1801
复制
发表时间：
2001-04
影响因子：
14.9
通讯作者：
Gan Wang;Zhimin Chen;Shijun Zhang;G. Wilson;Kai Jing
中科院分区：
文献类型：
--
作者：
Gan Wang;Zhimin Chen;Shijun Zhang;G. Wilson;Kai Jing
转录偶联修复（TCR）在清除活跃转录基因的DNA损伤中起着重要作用。据推测，TCR是修复非分裂细胞如神经元中DNA损伤的最重要机制。因此，异常TCR可能有助于许多年龄相关性和神经退行性疾病的发展。然而，TCR的分子机制尚未完全了解。寡核苷酸DNA三链体的形成提供了一个理想的系统来剖析TCR的分子机制，因为三链体可以以序列特异性的方式形成以抑制靶基因的转录。我们最近研究了HeLa细胞核提取物中三链体形成寡核苷酸（TFO）介导的TCR的分子机制。使用质粒构建体，我们证明了TFO介导的DNA修复活性的水平与HeLa细胞核提取物中质粒的转录水平直接相关。TF0介导的DNA修复活性进一步与转录相关，因为反应中rNTPs的存在对于HeLa细胞核提取物中AG30介导的DNA修复活性是必不可少的。在HeLa细胞核提取物中，使用免疫耗竭试验证明了包括TFIID、TFIIH、RNA聚合酶II和着色性干皮病A组（XPA）在内的单个组分参与三链体介导的TCR过程。重要的是，我们的研究还表明，XPC，一个参与全球基因组DNA修复的组件，参与AG30介导的DNA修复过程。本研究的结果为哺乳动物细胞TCR过程中的分子机制提供了重要的新认识。
Transcription-coupled repair (TCR) plays an important role in removing DNA damage from actively transcribed genes. It has been speculated that TCR is the most important mechanism for repairing DNA damage in non-dividing cells such as neurons. Therefore, abnormal TCR may contribute to the development of many age-related and neurodegenerative diseases. However, the molecular mechanism of TCR is not well understood. Oligonucleotide DNA triplex formation provides an ideal system to dissect the molecular mechanism of TCR since triplexes can be formed in a sequence-specific manner to inhibit transcription of target genes. We have recently studied the molecular mechanism of triplex-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extracts. Using plasmid constructs we demonstrate that the level of TFO-mediated DNA repair activity is directly correlated with the level of transcription of the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity was further linked with transcription since the presence of rNTPs in the reaction was essential for AG30-mediated DNA repair activity in HeLa nuclear extracts. The involvement of individual components, including TFIID, TFIIH, RNA polymerase II and xeroderma pigmentosum group A (XPA), in the triplex-mediated TCR process was demonstrated in HeLa nuclear extracts using immunodepletion assays. Importantly, our studies also demonstrated that XPC, a component involved in global genome DNA repair, is involved in the AG30-mediated DNA repair process. The results obtained in this study provide an important new understanding of the molecular mechanisms involved in the TCR process in mammalian cells.