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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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Tethering XPB to chromatin triggers the recruitment of downstream NER factors. (A) Schematic representation of XPB(1–782)-LacR-GFP and XPB(1–782)-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B, lanes 1–3) Proteins from whole cell extracts (15 µg) of U2OS17-expressing XPB proteins were resolved by SDS-PAGE followed by Western blotting using anti-XPB antibodies. (lanes 4–6) TFIIH from 300 µg of U2OS17 extracts was immunoprecipitated with an anti-p62 antibody and resolved by SDS-PAGE followed by Western blotting with an anti-XPB antibody. The asterisk indicates the endogenous XPB. (C–G) Recruitment of XPC (C), hHR23b (D), RFP-TTDA (E), Flag-XPA (F), and HA-XPF (G; Su et al., 2012) to tethered XPB-LacR-GFP (a–c). 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.
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fig3: Tethering XPB to chromatin triggers the recruitment of downstream NER factors. (A) Schematic representation of XPB(1–782)-LacR-GFP and XPB(1–782)-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B, lanes 1–3) Proteins from whole cell extracts (15 µg) of U2OS17-expressing XPB proteins were resolved by SDS-PAGE followed by Western blotting using anti-XPB antibodies. (lanes 4–6) TFIIH from 300 µg of U2OS17 extracts was immunoprecipitated with an anti-p62 antibody and resolved by SDS-PAGE followed by Western blotting with an anti-XPB antibody. The asterisk indicates the endogenous XPB. (C–G) Recruitment of XPC (C), hHR23b (D), RFP-TTDA (E), Flag-XPA (F), and HA-XPF (G; Su et al., 2012) to tethered XPB-LacR-GFP (a–c). 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.

Mentions: We subsequently tested the reciprocity of the recruitments that we observed by tethering TFIIH (XPB) to the LacO array (Fig. 3 A). To verify the functionality of the chimeric protein, we performed immunoprecipitation experiments using anti-p62, another TFIIH subunit, and observed a similar incorporation of XPB(1–782)-GFP and XPB(1–782)-LacR-GFP into TFIIH (Fig. 3 B). Furthermore, we observed colocalization of XPB(1–782)-LacR-GFP with XPC in a local UV irradiation experiment (Fig. S1 C). Also, TFIIH containing the XPB(1–782)-LacR-GFP was functional in a dual incision assay (unpublished data), showing that the LacR fusion did not inhibit XPB function.


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)

Tethering XPB to chromatin triggers the recruitment of downstream NER factors. (A) Schematic representation of XPB(1–782)-LacR-GFP and XPB(1–782)-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B, lanes 1–3) Proteins from whole cell extracts (15 µg) of U2OS17-expressing XPB proteins were resolved by SDS-PAGE followed by Western blotting using anti-XPB antibodies. (lanes 4–6) TFIIH from 300 µg of U2OS17 extracts was immunoprecipitated with an anti-p62 antibody and resolved by SDS-PAGE followed by Western blotting with an anti-XPB antibody. The asterisk indicates the endogenous XPB. (C–G) Recruitment of XPC (C), hHR23b (D), RFP-TTDA (E), Flag-XPA (F), and HA-XPF (G; Su et al., 2012) to tethered XPB-LacR-GFP (a–c). 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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig3: Tethering XPB to chromatin triggers the recruitment of downstream NER factors. (A) Schematic representation of XPB(1–782)-LacR-GFP and XPB(1–782)-GFP. For clarity, the sizes of the GFP (238 aa) and LacR (367 aa) were omitted. (B, lanes 1–3) Proteins from whole cell extracts (15 µg) of U2OS17-expressing XPB proteins were resolved by SDS-PAGE followed by Western blotting using anti-XPB antibodies. (lanes 4–6) TFIIH from 300 µg of U2OS17 extracts was immunoprecipitated with an anti-p62 antibody and resolved by SDS-PAGE followed by Western blotting with an anti-XPB antibody. The asterisk indicates the endogenous XPB. (C–G) Recruitment of XPC (C), hHR23b (D), RFP-TTDA (E), Flag-XPA (F), and HA-XPF (G; Su et al., 2012) to tethered XPB-LacR-GFP (a–c). 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.
Mentions: We subsequently tested the reciprocity of the recruitments that we observed by tethering TFIIH (XPB) to the LacO array (Fig. 3 A). To verify the functionality of the chimeric protein, we performed immunoprecipitation experiments using anti-p62, another TFIIH subunit, and observed a similar incorporation of XPB(1–782)-GFP and XPB(1–782)-LacR-GFP into TFIIH (Fig. 3 B). Furthermore, we observed colocalization of XPB(1–782)-LacR-GFP with XPC in a local UV irradiation experiment (Fig. S1 C). Also, TFIIH containing the XPB(1–782)-LacR-GFP was functional in a dual incision assay (unpublished data), showing that the LacR fusion did not inhibit XPB function.

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.

Show MeSH
Related in: MedlinePlus