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. 2004 Apr 30;279(18):19074-83.
doi: 10.1074/jbc.M312611200. Epub 2004 Feb 23.

Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors

Angels Tapias  1 , Jerome Auriol, Diane Forget, Jacqueline H Enzlin, Orlando D Schärer, Frederic Coin, Benoit Coulombe, Jean-Marc Egly
Affiliations

Ordered conformational changes in damaged DNA induced by nucleotide excision repair factors

Angels Tapias et al. J Biol Chem. .

Abstract

In response to genotoxic attacks, cells activate sophisticated DNA repair pathways such as nucleotide excision repair (NER), which consists of damage removal via dual incision and DNA resynthesis. Using permanganate footprinting as well as highly purified factors, we show that NER is a dynamic process that takes place in a number of successive steps during which the DNA is remodeled around the lesion in response to the various NER factors. XPC/HR23B first recognizes the damaged structure and initiates the opening of the helix from position -3 to +6. TFIIH is then recruited and, in the presence of ATP, extends the opening from position -6 to +6; it also displaces XPC downstream from the lesion, thereby providing the topological structure for recruiting XPA and RPA, which will enlarge the opening. Once targeted by XPG, the damaged DNA is further melted from position -19 to +8. XPG and XPF/ERCC1 endonucleases then cut the damaged DNA at the limit of the opened structure that was previously "labeled" by the positioning of XPC/HR23B and TFIIH.

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Figures

Fig. 1
Fig. 1. NER factors used in dual incision and damaged DNA opening
A, the purified NER factors XPC, HR23B, XPC/HR23B, TFIIH, RPA, XPG, and XPF/ERCC1 were stained with Coomassie Blue. B, dual incision with the purified factors. Repair factors were individually omitted as shown in lanes 2–7. In lane 1, the repair reaction was performed in the presence of all NER factors. nt, nucleotide. C, KMnO4 modifications. The nucleotides marked with asterisks match up sequentially with the arrows that point to bands of interest. The damaged strand (left panel, lanes 1–3) and undamaged strand probes (right panel, lanes 4–7) were incubated with ROS containing XPC/HR23B, TFIIH, XPA, RPA, and XPG. When indicated, ROS lacks either TFIIH (lanes 2 and 6) or XPC/HR23B (lane 7). Lane 1, bovine serum albumin (100 μg). An autoradiography of the gel is shown on the far right with dideoxy sequencing reaction A, G, C, and T ladders as markers. Pt, position 0.
Fig. 2
Fig. 2. Contribution of the NER factors to the DNA opening
A, XPC/HR23B and XPF/ERCC1 target the damaged DNA. Each NER factor was added separately to the damaged strand probe and tested for KMnO4 modification as indicated by arrows at the right of the panel. Bands corresponding to the 3′ incision are indicated by an arrowhead. B, combinations of the NER factors (except XPC/HR23B) as indicated at the top of the panel. C, XPC (40 ng) and HR23B (40 ng), either alone or in combination (lanes 5 and 9), or the purified XPC/HR23B complex from the co-infected extract (lanes 2 and 6) were tested for KMnO4 modifications (left panel) in the absence (lanes 2–5) or presence of XPA, RPA, TFIIH, and XPG (lanes 6–9). Dual incision in the presence of HR23B (10 – 40 ng) and XPC (10, 40 and 160 ng) either alone or in combination (XPC, 10 ng; XPC together with 50, 100, and 150 ng of HR23B). Pt, position 0; nt, nucleotide.
Fig. 3
Fig. 3. Ordered remodeling of the damaged DNA upon NER factor recruitment
A, KMnO4 modification in which NER factors were individually omitted. B, ATP induces DNA unwinding by TFIIH (lanes 1–13). Recruitment of XPA, RPA, and XPG to the XPC/HR23B-TFIIH-damaged DNA complex is depicted (lanes 14–19); the addition of XPF/ERCC1 to ROS is shown (lanes 20–21). C, RPA does not modify the KMnO4 footprinting of the undamaged strand already targeted by XPC/HR23B and TFIIH. The nucleotides marked with asterisks match up sequentially with the arrows that point to bands of interest. Bands corresponding to the 3′ incision are indicated by an arrowhead. Pt, position 0.
Fig. 4
Fig. 4. XPG and XPF/ERCC1 extend the DNA opening and excise the damaged oligonucleotide
A, time course of DNA incision in which XPC/HR23B, TFIIH, XPA, RPA and damaged strand probe were incubated in the presence or absence of either XPG (lanes 1–9) or XPF/ERCC1 (lanes 10–18). Kinetics of KMnO4 modification (B) and DNA incision (C) upon the addition of XPF/ERCC1 to the five NER factors, including XPG. The 3′ and 5′ incisions are indicated on each side of the panel. The nucleotides marked with asterisks match up sequentially with the arrows that point to bands of interest. Pt, position 0.
Fig. 5
Fig. 5. Mutant XPF/ERCC1 enhances DNA opening
A, dual incision assays using either XPF/ERCC1 wild type (wt) (12, 6, or 3 ng), XPF-D676A and XPF-D720A/ERCC1 (50 and 18 ng), in addition to the five NER factors. nt, nucleotide. B, KMnO4 modifications (left panel) and DNA incisions (right panel) in the presence of XPF-D676A, -D720A, and -wt/ERCC1 as indicated at the top of the panel. KMnO4-induced cuts as well as incisions are indicated on each side of the panel. The nucleotides marked with asterisks match up sequentially with the arrows that point to bands of interest. Pt, position 0.
Fig. 6
Fig. 6. Photo Cross-linking of XPC and XPB to specific positions along the damaged DNA
A, four cisplatinated photoprobes (−23/−20, −8/−7, +12/+15, and +21/+22) that place two photoreactive nucleotides AB-dUMP (U) in juxtaposition to radiolabeled nucleotides (*) at specific locations along the damaged DNA (Pt, position 0) were synthesized and used in the photo cross-linking experiments. B, XPC/HR23B alone or XPC/HR23B and TFIIH in the presence of 1 mM ATP were incubated with each photoprobe, and the complexes were UV irradiated and processed to determine the polypeptides that cross-link to the various photoprobes. The position of XPC, XPB, and the molecular weight markers are indicated. C, relative intensities of the XPC and XPB cross-linking signals in three independent experiments using our four photoprobes when both XPC/HR23B and TFIIH were present in the reactions. S.D. is indicated.
Fig. 7
Fig. 7. Opening of the DNA around the Pt-GTG crosslink by the NER factors
A, opening around the Pt-GTG during NER, as evidenced by reactivity to KMnO4, is depicted by placement of these residues above or below the axis of the duplex sequence. The position of the incision sites by XPG and XPF/ERCC1 endo-nucleases are indicated by arrows. The bottom part of the figure summarizes the photo cross-linking data with XPC/HR23B, either alone (open box) or in the presence of TFIIH and ATP (grey box for XPC, black box for XPB). B, a model representing the relocalization of XPC in the presence of TFIIH and the various steps leading to the removal of the damaged oligonucleotide. Each step is characterized by an increase in the remodeling of damaged DNA that is progressively opened by the repair machinery. In the absence of TFIIH, XPC approaches the damaged DNA from nucleotides −20 to +20, the contact at positions −8/−7 being the strongest. In the presence of TFIIH, XPC cross-links strongly to positions −23/−20 and weakly to positions −8/−7, but does not cross-link to positions on the 3′ end of the cisplatin adduct. The fully open complex (−19 to +8) is used as a template for incision by XPF/ERCC1 and XPG as indicated by arrows.
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