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Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin.

Ziani S, Nagy Z, Alekseev S, Soutoglou E, Egly JM, Coin F - J. Cell Biol. (2014)

Bottom Line: We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC.TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients.More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment.

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Affiliation: Department of Functional Genomics and Cancer, Equipe Labellisée Ligue 2014; and Department of Development Biology and Stem Cells, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/University of Strasbourg, 67404 Illkirch Cedex, Communauté urbaine de Strasbourg, France.

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The N-terminal domain of TTDA recruits XPA to damaged DNA. (A) Schematic representation of the TTDA(1–71)-GFP, TTDA(1–71)-LacR-GFP, and TTDA(15–71)-LacR-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B) Proteins from whole cell extracts (15 µg) of U2OS17-expressing TTDA proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (C–E) Recruitment of XPC (C), XPB (D), and Flag-XPA (E) to either tethered TTDA(1–71)-LacR-GFP or TTDA(15–71)-LacR-GFP as indicated. The values on the graphs represent the percentage of colocalization of each NER factor with GFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (F) Flag-TTDA(1–71) or Flag-TTDA(15–71) were expressed in E. coli and immunoprecipitated with anti-flag antibody–covered beads. After washes, the beads were incubated with bacterial extracts expressing XPD, p52, or XPA. Pull-down fractions were washed, resolved by SDS-PAGE, and immunoblotted with anti-XPD, p52, and XPA antibodies (top) or anti-Flag antibody (bottom). (G) 10 and 30 ng of either flag-TTDA(1–71) or flag-TTDA(15–71) were tested in a dual incision assay (NER) containing the recombinant XPC-HR23b, recombinant TFIIH lacking TTDA (IIH9; Coin et al., 2006), XPA, RPA, XPG, ERCC1-XPF factors, and a closed circular plasmid containing a single 1,3-intra strand d(GpTpG) cisplatin-DNA cross-link (Pt-DNA) as a template. Sizes of the incision products are indicated. (H) Immobilized biotinylated damaged DNA was incubated with XPC/hHR23B, IIH9, XPA, and either flag-TTDA(1–71) or flag-TTDA(15–71) as indicated (PreInc) for 15 min at 30°C with ATP. DNA was washed, supplemented with XPC/hHR23b, purified TFIIH from HeLa, RPA, ERCC1-XPF, and XPG without XPA, and subjected to dual incision assay. In lane 3, all the NER factors were added in a single step with immobilized damaged DNA and subjected to dual incision.
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fig5: The N-terminal domain of TTDA recruits XPA to damaged DNA. (A) Schematic representation of the TTDA(1–71)-GFP, TTDA(1–71)-LacR-GFP, and TTDA(15–71)-LacR-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B) Proteins from whole cell extracts (15 µg) of U2OS17-expressing TTDA proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (C–E) Recruitment of XPC (C), XPB (D), and Flag-XPA (E) to either tethered TTDA(1–71)-LacR-GFP or TTDA(15–71)-LacR-GFP as indicated. The values on the graphs represent the percentage of colocalization of each NER factor with GFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (F) Flag-TTDA(1–71) or Flag-TTDA(15–71) were expressed in E. coli and immunoprecipitated with anti-flag antibody–covered beads. After washes, the beads were incubated with bacterial extracts expressing XPD, p52, or XPA. Pull-down fractions were washed, resolved by SDS-PAGE, and immunoblotted with anti-XPD, p52, and XPA antibodies (top) or anti-Flag antibody (bottom). (G) 10 and 30 ng of either flag-TTDA(1–71) or flag-TTDA(15–71) were tested in a dual incision assay (NER) containing the recombinant XPC-HR23b, recombinant TFIIH lacking TTDA (IIH9; Coin et al., 2006), XPA, RPA, XPG, ERCC1-XPF factors, and a closed circular plasmid containing a single 1,3-intra strand d(GpTpG) cisplatin-DNA cross-link (Pt-DNA) as a template. Sizes of the incision products are indicated. (H) Immobilized biotinylated damaged DNA was incubated with XPC/hHR23B, IIH9, XPA, and either flag-TTDA(1–71) or flag-TTDA(15–71) as indicated (PreInc) for 15 min at 30°C with ATP. DNA was washed, supplemented with XPC/hHR23b, purified TFIIH from HeLa, RPA, ERCC1-XPF, and XPG without XPA, and subjected to dual incision assay. In lane 3, all the NER factors were added in a single step with immobilized damaged DNA and subjected to dual incision.

Mentions: The aforementioned data show that the presence of TTDA in TFIIH on the LacO array correlates with the recruitment of XPA and XPF (Figs. 1 and 3), pinpointing to a crucial role of this small polypeptide in the completion of the PInC. To study this hypothesis, we constructed the TTDA(1–71)-LacR-GFP fusion protein (Fig. 5, A and B) that colocalized with XPC on local UV-induced DNA damage (Fig. S1 E). Prolonged immobilization of TTDA(1–71)-LacR-GFP in U2OS17 cells induced the recruitment of XPB, XPA, and XPF but not that of XPC or hHR23b (Fig. 5, C–E; and Fig. S2 B).


Sequential and ordered assembly of a large DNA repair complex on undamaged chromatin.

Ziani S, Nagy Z, Alekseev S, Soutoglou E, Egly JM, Coin F - J. Cell Biol. (2014)

The N-terminal domain of TTDA recruits XPA to damaged DNA. (A) Schematic representation of the TTDA(1–71)-GFP, TTDA(1–71)-LacR-GFP, and TTDA(15–71)-LacR-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B) Proteins from whole cell extracts (15 µg) of U2OS17-expressing TTDA proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (C–E) Recruitment of XPC (C), XPB (D), and Flag-XPA (E) to either tethered TTDA(1–71)-LacR-GFP or TTDA(15–71)-LacR-GFP as indicated. The values on the graphs represent the percentage of colocalization of each NER factor with GFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (F) Flag-TTDA(1–71) or Flag-TTDA(15–71) were expressed in E. coli and immunoprecipitated with anti-flag antibody–covered beads. After washes, the beads were incubated with bacterial extracts expressing XPD, p52, or XPA. Pull-down fractions were washed, resolved by SDS-PAGE, and immunoblotted with anti-XPD, p52, and XPA antibodies (top) or anti-Flag antibody (bottom). (G) 10 and 30 ng of either flag-TTDA(1–71) or flag-TTDA(15–71) were tested in a dual incision assay (NER) containing the recombinant XPC-HR23b, recombinant TFIIH lacking TTDA (IIH9; Coin et al., 2006), XPA, RPA, XPG, ERCC1-XPF factors, and a closed circular plasmid containing a single 1,3-intra strand d(GpTpG) cisplatin-DNA cross-link (Pt-DNA) as a template. Sizes of the incision products are indicated. (H) Immobilized biotinylated damaged DNA was incubated with XPC/hHR23B, IIH9, XPA, and either flag-TTDA(1–71) or flag-TTDA(15–71) as indicated (PreInc) for 15 min at 30°C with ATP. DNA was washed, supplemented with XPC/hHR23b, purified TFIIH from HeLa, RPA, ERCC1-XPF, and XPG without XPA, and subjected to dual incision assay. In lane 3, all the NER factors were added in a single step with immobilized damaged DNA and subjected to dual incision.
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Related In: Results  -  Collection

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fig5: The N-terminal domain of TTDA recruits XPA to damaged DNA. (A) Schematic representation of the TTDA(1–71)-GFP, TTDA(1–71)-LacR-GFP, and TTDA(15–71)-LacR-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B) Proteins from whole cell extracts (15 µg) of U2OS17-expressing TTDA proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (C–E) Recruitment of XPC (C), XPB (D), and Flag-XPA (E) to either tethered TTDA(1–71)-LacR-GFP or TTDA(15–71)-LacR-GFP as indicated. The values on the graphs represent the percentage of colocalization of each NER factor with GFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (F) Flag-TTDA(1–71) or Flag-TTDA(15–71) were expressed in E. coli and immunoprecipitated with anti-flag antibody–covered beads. After washes, the beads were incubated with bacterial extracts expressing XPD, p52, or XPA. Pull-down fractions were washed, resolved by SDS-PAGE, and immunoblotted with anti-XPD, p52, and XPA antibodies (top) or anti-Flag antibody (bottom). (G) 10 and 30 ng of either flag-TTDA(1–71) or flag-TTDA(15–71) were tested in a dual incision assay (NER) containing the recombinant XPC-HR23b, recombinant TFIIH lacking TTDA (IIH9; Coin et al., 2006), XPA, RPA, XPG, ERCC1-XPF factors, and a closed circular plasmid containing a single 1,3-intra strand d(GpTpG) cisplatin-DNA cross-link (Pt-DNA) as a template. Sizes of the incision products are indicated. (H) Immobilized biotinylated damaged DNA was incubated with XPC/hHR23B, IIH9, XPA, and either flag-TTDA(1–71) or flag-TTDA(15–71) as indicated (PreInc) for 15 min at 30°C with ATP. DNA was washed, supplemented with XPC/hHR23b, purified TFIIH from HeLa, RPA, ERCC1-XPF, and XPG without XPA, and subjected to dual incision assay. In lane 3, all the NER factors were added in a single step with immobilized damaged DNA and subjected to dual incision.
Mentions: The aforementioned data show that the presence of TTDA in TFIIH on the LacO array correlates with the recruitment of XPA and XPF (Figs. 1 and 3), pinpointing to a crucial role of this small polypeptide in the completion of the PInC. To study this hypothesis, we constructed the TTDA(1–71)-LacR-GFP fusion protein (Fig. 5, A and B) that colocalized with XPC on local UV-induced DNA damage (Fig. S1 E). Prolonged immobilization of TTDA(1–71)-LacR-GFP in U2OS17 cells induced the recruitment of XPB, XPA, and XPF but not that of XPC or hHR23b (Fig. 5, C–E; and Fig. S2 B).

Bottom Line: We also revealed that the recruitment of the TFIIH subunit TTDA, involved in trichothiodystrophy group A disorder (TTD-A), was key in the completion of the PInC.TTDA recruits XPA through its first 15 amino acids, depleted in some TTD-A patients.More generally, these results show that proteins forming large nuclear complexes can be recruited sequentially on chromatin in the absence of their natural DNA target and with no reciprocity in their recruitment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Functional Genomics and Cancer, Equipe Labellisée Ligue 2014; and Department of Development Biology and Stem Cells, Institut de Génétique et de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/University of Strasbourg, 67404 Illkirch Cedex, Communauté urbaine de Strasbourg, France.

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Related in: MedlinePlus