Detection and determination of oligonucleotide tripler 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 tripler forma tion provides an ideal system to dissect the molecular mechanism of TCR sin ce triplexes can be formed in a sequence-specific manner to inhibit transcr iption of target genes. We have recently studied the molecular mechanism of tripler-forming oligonucleotide (TFO)-mediated TCR in HeLa nuclear extract s, Using plasmid constructs we demonstrate that the level of TFO-mediated D NA repair activity is directly correlated with the level of transcription o f the plasmid in HeLa nuclear extracts. TFO-mediated DNA repair activity wa s further linked with transcription since the presence of rNTPs in the reac tion was essential for AG30-mediated DNA repair activity in HeLa nuclear ex tracts. The involvement of individual components, including TFIID, TFIIH, R NA polymerase II and xeroderma pigmentosum group A (XPA), in the tripler-me diated TCR process was demonstrated in HeLa nuclear extracts using immunode pletion assays. importantly, our studies also demonstrated that XPC, a comp onent involved in global genome DNA repair, is involved in the AG30-mediate d DNA repair process. The results obtained in this study provide an importa nt new understanding of the molecular mechanisms involved in the TCR proces s in mammalian cells.