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

Abstract

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.

Figure 1

Structure of plasmids pUSAG15 and pUSAG16. pUSAG15 was constructed by cloning a 177 bp supF gene DNA fragment containing a 30 bp homopurine triplex-binding site from pSupFG1 into the HindIII site of vector pGL3-Basic. pUSAG16 was constructed by inserting the 177 bp supF gene DNA fragment into the HindIII site of vector pGL3-Control. TBS, triplex-binding sequence; H, restriction enzyme HindIII digestion site; B, restriction enzyme BamHI digestion site.

Figure 2

TFO AG30-mediated DNA repair synthesis of pUSAG15 and pUSAG16 in HeLa cell nuclear extracts. The supercoiled plasmid DNA was preincubated with oligonucleotide at 37°C for 2 h in triplex-binding buffer (10 mM Tris pH 7.5, 1 mM spermidine, 20 mM MgCl₂) for triplex formation. Then the plasmid DNA (1 µg) was incubated with HeLa nuclear extracts supplemented with dATP, dGTP, dTTP and [α-³²P]dCTP at 30°C for 2 h. The plasmid DNAs were linearized with XhoI restriction enzyme and analyzed by agarose gel electrophoresis using a 0.8% gel. (A) Visualization of the plasmid DNA by ethidium bromide staining. (B) Autoradiogram of the same gel showing labeled nucleotide incorporation indicative of DNA repair synthesis. (C) Quantification of incorporation of [α-³²P]dCTP into plasmid DNA. The amount of incorporated [α-³²P]dCTP in pUSAG15–AG30 was taken as 100%. Incorporation of [α-³²P]dCTP in other reactions was calculated as a percentage of pUSAG15–AG30. The data are means obtained from four individual experiments.

Figure 3

Detection of transcription of both pUSAG15 and pUSAG16 in HeLa nuclear extract. The in vitro transcription reactions with pUSAG15 and pUSAG16 were performed in HeLa nuclear extracts. (A) Detection of RNA transcripts synthesized from pUSAG15 and pUSAG16 plasmid DNA in HeLa nuclear extract. Lane 1, pUSAG15; lane 2, pUSAG16. (B) Reverse transcription assay to determine the initiation sites of RNA transcripts synthesized from pUSAG15 and pUSAG16 plasmid DNA in HeLa nuclear extract. Lane 1, no DNA control; lane 2, pUSAG15; lane 3, pUSAG16 (the arrows indicate specific RNA transcripts initiated from the SV40 promoter).

Figure 4

Involvement of individual components in TFO AG30 triplex-mediated DNA repair synthesis of pUSAG15. Individual components were immunodepleted from HeLa nuclear extracts using antibodies against specific components. (A) Visualization of plasmid DNA by ethidium bromide staining. (B) Autoradiogram of the same gel showing labeled nucleotide incorporation indicative of DNA repair synthesis. (C) Quantification of incorporated [α-³²P]dCTP in plasmid DNA. Lane 1, CT30 + HeLa nuclear extract; lane 2, AG30 + HeLa nuclear extract; lane 3, AG30 + XPA-depleted HeLa nuclear extract; lane 4, AG30 + TFIID-depleted HeLa nuclear extract; lane 5, AG30 + TFIIH-depleted HeLa nuclear extract; lane 6, AG30 + Pol II-depleted HeLa nuclear extract; lane 7, AG30 + rNTP-lacking HeLa nuclear extract. The data are means obtained from four individual experiments.

Figure 5

TFO AG30 triplex-mediated DNA repair of pUSAG15 in nuclear extracts prepared from XPA, XPC and CSA cells. Lane 1, CT30 + NF nuclear extract (NF); lane 2, AG30 + NF nuclear extract (NF); lane 3, AG30 + XPA nuclear extract (XPA_N); lane 4, AG30 + XPA_N + purified XPA protein (XPA_P); lane 5, AG30 + XPC nuclear extract (XPC_N); lane 6, AG30 + XPC_N + purified XPC protein (XPC_P); lane 7, AG30 + XPA_N + XPC_P; lane 8, AG30 + XPC_N + XPA_P; lane 9, AG30 + CSA nuclear extract (CSA_N); lane 10, AG30 + CSA_N + XPC_N.