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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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Defining TFIIH- and hHR23b-interacting domains in XPC. (A) Schematic representation of the wild-type and mutant XPC constructs. 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 wild-type or mutant XPC proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibody. (C and D) Recruitment of hHR23b (C) and XPB (D) to tethered GFP-LacR-XPC(1–940) (a–c), GFP-LacR-XPC(1–579) (d–f), GFP-LacR-XPC(1–200) (g–i), or GFP-LacR-XPC(200−940) (j–l). 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. (E) After transfection of GFP-LacR-XPC(1–940) or GFP-LacR-XPC(200–940), XP-C cells were locally UV irradiated (150 J/m2), fixed 15 min later, and stained with antibodies raised against GFP, XPB, and UV-induced CPD. The values on the graphs represent the percentage of colocalization of XPB with GFP on the local UV array based on three independent experiments with SD. Arrowheads indicate locally irradiated areas. (F) After transfection of GFP-LacR, GFP-LacR-XPC(1–940), or GFP-LacR-XPC(200–940) (GFP), XP-C cells were locally UV irradiated (150 J/m2) and repair synthesis was analyzed by EdU incorporation at DNA damage (CPD). Arrowheads indicate locally irradiated areas.
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fig2: Defining TFIIH- and hHR23b-interacting domains in XPC. (A) Schematic representation of the wild-type and mutant XPC constructs. 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 wild-type or mutant XPC proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibody. (C and D) Recruitment of hHR23b (C) and XPB (D) to tethered GFP-LacR-XPC(1–940) (a–c), GFP-LacR-XPC(1–579) (d–f), GFP-LacR-XPC(1–200) (g–i), or GFP-LacR-XPC(200−940) (j–l). 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. (E) After transfection of GFP-LacR-XPC(1–940) or GFP-LacR-XPC(200–940), XP-C cells were locally UV irradiated (150 J/m2), fixed 15 min later, and stained with antibodies raised against GFP, XPB, and UV-induced CPD. The values on the graphs represent the percentage of colocalization of XPB with GFP on the local UV array based on three independent experiments with SD. Arrowheads indicate locally irradiated areas. (F) After transfection of GFP-LacR, GFP-LacR-XPC(1–940), or GFP-LacR-XPC(200–940) (GFP), XP-C cells were locally UV irradiated (150 J/m2) and repair synthesis was analyzed by EdU incorporation at DNA damage (CPD). Arrowheads indicate locally irradiated areas.

Mentions: To obtain insights into the formation of the PInC in the absence of DNA damage, we used the LacO/LacR reporter system (Tumbar et al., 1999; Soutoglou and Misteli, 2008). The full-length human GFP-XPC(1–940) construct (Bernardes de Jesus et al., 2008) was fused to the LacR (Fig. 1 A, top). In a local UV irradiation experiment (Coin et al., 2006), GFP-LacR-XPC(1–940) colocalized with XPB on UV-C–induced DNA damage (Fig. S1, A and B), showing its functionality (see also Fig. 2, E and F).


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)

Defining TFIIH- and hHR23b-interacting domains in XPC. (A) Schematic representation of the wild-type and mutant XPC constructs. 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 wild-type or mutant XPC proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibody. (C and D) Recruitment of hHR23b (C) and XPB (D) to tethered GFP-LacR-XPC(1–940) (a–c), GFP-LacR-XPC(1–579) (d–f), GFP-LacR-XPC(1–200) (g–i), or GFP-LacR-XPC(200−940) (j–l). 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. (E) After transfection of GFP-LacR-XPC(1–940) or GFP-LacR-XPC(200–940), XP-C cells were locally UV irradiated (150 J/m2), fixed 15 min later, and stained with antibodies raised against GFP, XPB, and UV-induced CPD. The values on the graphs represent the percentage of colocalization of XPB with GFP on the local UV array based on three independent experiments with SD. Arrowheads indicate locally irradiated areas. (F) After transfection of GFP-LacR, GFP-LacR-XPC(1–940), or GFP-LacR-XPC(200–940) (GFP), XP-C cells were locally UV irradiated (150 J/m2) and repair synthesis was analyzed by EdU incorporation at DNA damage (CPD). Arrowheads indicate locally irradiated areas.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4151144&req=5

fig2: Defining TFIIH- and hHR23b-interacting domains in XPC. (A) Schematic representation of the wild-type and mutant XPC constructs. 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 wild-type or mutant XPC proteins were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibody. (C and D) Recruitment of hHR23b (C) and XPB (D) to tethered GFP-LacR-XPC(1–940) (a–c), GFP-LacR-XPC(1–579) (d–f), GFP-LacR-XPC(1–200) (g–i), or GFP-LacR-XPC(200−940) (j–l). 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. (E) After transfection of GFP-LacR-XPC(1–940) or GFP-LacR-XPC(200–940), XP-C cells were locally UV irradiated (150 J/m2), fixed 15 min later, and stained with antibodies raised against GFP, XPB, and UV-induced CPD. The values on the graphs represent the percentage of colocalization of XPB with GFP on the local UV array based on three independent experiments with SD. Arrowheads indicate locally irradiated areas. (F) After transfection of GFP-LacR, GFP-LacR-XPC(1–940), or GFP-LacR-XPC(200–940) (GFP), XP-C cells were locally UV irradiated (150 J/m2) and repair synthesis was analyzed by EdU incorporation at DNA damage (CPD). Arrowheads indicate locally irradiated areas.
Mentions: To obtain insights into the formation of the PInC in the absence of DNA damage, we used the LacO/LacR reporter system (Tumbar et al., 1999; Soutoglou and Misteli, 2008). The full-length human GFP-XPC(1–940) construct (Bernardes de Jesus et al., 2008) was fused to the LacR (Fig. 1 A, top). In a local UV irradiation experiment (Coin et al., 2006), GFP-LacR-XPC(1–940) colocalized with XPB on UV-C–induced DNA damage (Fig. S1, A and B), showing its functionality (see also Fig. 2, E and F).

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