Initiation of DNA repair mediated by a stalled RNA polymerase IIO

Abstract

The transcription-coupled repair (TCR) pathway preferentially repairs DNA damage located in the transcribed strand of an active gene. To gain insight into the coupling mechanism between transcription and repair, we have set up an in vitro system in which we isolate an elongating RNA pol IIO, which is stalled in front of a cisplatin adduct. This immobilized RNA pol IIO is used as 'bait' to sequentially recruit TFIIH, XPA, RPA, XPG and XPF repair factors in an ATP-dependent manner. This RNA pol IIO/repair complex allows the ATP-dependent removal of the lesion only in the presence of CSB, while the latter does not promote dual incision in an XPC-dependent nucleotide excision repair reaction. In parallel to the dual incision, the repair factors also allow the partial release of RNA pol IIO. In this 'minimal TCR system', the RNA pol IIO can effectively act as a loading point for all the repair factors required to eliminate a transcription-blocking lesion.

Figure 1

A template for both transcription and DNA repair reactions. (A) The transcription/repair template (Cax-Pt) contains a single cisplatin adduct (GTG, Pt) at position +105 nt in the transcribed strand downstream from the adenovirus major late promoter. The TATA box is represented by a triangle and the start site +1 by a bent arrow. The positions of the restriction enzymes sites are indicated. (B) Coomassie staining of highly purified transcription and repair factors. (C) Transcription on Cax (lanes 1–5) and Cax-Pt (lanes 6–10) was performed using either RTS or WCE/XPC as indicated at the top of the panel. Full length (328 nt) or prematurated stopped transcripts (105 nt) were resolved on 8% urea/PAGE. The Addition of α-amanitin is indicated. (D) Dual incision on Cax-Pt was carried out with RIS in either the presence of (lane 1) or the absence of XPC (RISΔXPC; lane 2), WCE/Hela (lane 3) or WCE/XPC (lane 4) supplemented with XPC (lane 5). Dual incision is indicated by the occurrence of a 26–34-nucleotides excision products.

Figure 2

Recruitment of NER factors onto the stalled RNA pol IIO. (A) Western blots of transcription complexes on immobilized DNA before (PIC, lane 1) and after (EC, lanes 2 and 3) the addition of nucleotide triphosphates. Immobilized protein complexes were washed either at 50 mM KCl (PIC-50 and EC-50) or at 400 mM KCl (EC-400). (B) Dual incision reaction on the immobilized Cax-Pt transcribed or not, and cut by ClaI. (C) Transcription reactions on Cax-Pt (lane 1) were further incubated with TFIIS or CSB (lanes 2 and 3). Arrows indicate the different lengths of RNA transcripts. (D) Immobilized ClaI-cut Cax-Pt (lane 1) as well as ClaI-cut EC-400 complex (lanes 2 and 3) were incubated with XPC. Following a second wash, these templates were tested in a dual incision reaction in which XPC is omitted. Fgt-Pt competitor damaged DNA (lane 2) is added where indicated. Complete NER (lane 4) is used as a control. (E) Immobilized Cax-Pt (lanes 1 and 4) or ClaI-cut EC-400 containing the stalled RNA pol IIO (lanes 2 and 3, 5–8) were pretreated with CIP (as indicated), and further incubated with either WCE/XPC or WCE/XPCΔCSB in the presence of ATP and CSB as indicated. Proteins remaining bound to the immobilized Cax-Pt template were next analyzed by Western blots. (F) Immobilized Cax-Pt (lanes 1) or ClaI-cut EC-400 (lanes 2–6) were treated as indicated at the top of the panel similarly to (E); the RNA pol IIO and CSB remaining on the immobilized Cax-Pt template were next analyzed by Western blots. (G) ClaI-cut Cax-Pt (lane 1) or ClaI-cut EC-400 complex (lane 2) were incubated with RISΔXPC, the dual incision system in which XPC is lacking. Following washes at 50 mM KCl, the remaining bound proteins were analyzed by Western blot.

Figure 3

RNA pol IIO/CSB-mediated incision. (A) EC-400 was incubated with RISΔXPC and increasing amounts of CSB, and subjected to a dual incision assay. Quantification was made as described in Materials and methods. A graphic depicts the relative intensity of each signal. (B) Dual incision assays were performed on EC-400, which was incubated with different combinations of NER factors and CSB as indicated. (C) EC-400 transcription complex (lanes 1–12) and Cax-Pt (lanes 13–15) pretreated or not with CIP (lanes 5 and 6) were incubated with RISΔXPC (lanes 2–15) and with either wild-type CSB (lanes 3–6, 9 and 15), mutated CSBΔ440, CSBΔ378, CSBR670W (lanes 10–12) or XPC (lane 13), and subjected to a 3′-incision primer extension assay. The position and scans of sensitive bands relative to cisplatin lesion are denoted by asterisks and indicated at the right of the gel. (D) Dual incisions were performed on the untranscribed Cax-Pt with RISΔXPC in the presence (lanes 1, 3–11) or the absence of XPC (lanes 2, 12 and 13), with either a limiting amount of TFIIH (lanes 3–5) or XPG (lanes 9–11). CSB was added in the reaction where indicated. The relative amounts of TFIIH and XPG are indicated by thick boxes (saturating amounts) and thin boxes (limiting amounts).

Figure 4

Sequential assembly of NER factors onto RNA pol IIO. (A) Scheme depicting the reaction. Cax-Pt was transcribed and then cut by ClaI to form the ClaI-cut EC-400, which was further incubated with different combinations of four repair factors (the omitted factor is referred to by Δ in each lane), or with different combinations of indicated NER factors. After soft washes (50 mM KCl), the recruited proteins were analyzed by Western blots (B) and (F) or by a functional dual incision complementation assay (see text for details). (C–E) Either RNA pol IIO (B) or RNA synthesis levels (C), (D) and (E) are used as a loading control.

Figure 5

NER factors-mediated release of RNA pol IIO. (A–E) EC-400 were incubated for 30 min at 30°C, either alone or together with NER factors and CSB as indicated at the top of each panel. The removal of RNA pol IIO from the DNA template in the supernatant was further analyzed by Western blots. (B) EC-400 was incubated with indicated factors in the absence or the presence of increasing amounts of ATP. IIH/XPB-f99s was also used (E). Quantification was made as described in Materials and methods. A graphic at the bottom of each panel depicts the relative intensity of each signal.