Wang G, Chen Z, Zhang S, Wilson G L, Jing K
Department of Cell Biology and Neuroscience, University of South Alabama College of Medicine, 307 University Boulevard, MSB 2312, Mobile, AL 36688-0002, USA.
Nucleic Acids Res. 2001 Apr 15;29(8):1801-7. doi: 10.1093/nar/29.8.1801.
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.
转录偶联修复（TCR）在去除活跃转录基因中的DNA损伤方面发挥着重要作用。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，TCR异常可能导致许多与年龄相关的疾病和神经退行性疾病的发生。然而，TCR的分子机制尚未完全清楚。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，从而抑制靶基因的转录。我们最近在HeLa细胞核提取物中研究了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修复活性水平与HeLa细胞核提取物中质粒的转录水平直接相关。TFO介导的DNA修复活性进一步与转录相关，因为反应中rNTP的存在对于HeLa细胞核提取物中AG30介导的DNA修复活性至关重要。使用免疫耗竭试验在HeLa细胞核提取物中证明了包括TFIID、TFIIH、RNA聚合酶II和A型着色性干皮病（XPA）在内的各个组分参与了三链体介导的TCR过程。重要的是，我们的研究还表明，参与全基因组DNA修复的组分XPC也参与了AG30介导的DNA修复过程。本研究获得的结果为哺乳动物细胞中TCR过程所涉及的分子机制提供了重要的新认识。
