Detection and determination of oligonucleotide triplex formation-mediated transcription-coupled DNA repair in HeLa nuclear extracts.

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.