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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 DDB2 to chromatin recruits XPC through its N- and C-terminal extremities. (A, left) Schematic representation of YFP-DDB2(1–427) and YFP-LacR-DDB2(1–427). For clarity, the sizes of the YFP (238 aa) and LacR (367 aa) were omitted. (right) Proteins from whole cell extracts (15 µg) of U2OS17 expressing either YFP-DDB2(1–427) or YFP-LacR-DDB2(1–427) were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (B–D) Recruitment of XPC (B), XPB (C), or flag-XPA (D) to tethered YFP-LacR-DDB2(1–427). The values on the graphs represent the percentage of colocalization of each NER factor with YFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (E) Schematic representation of wild-type and deleted flag-XPC constructs. (F) Recruitment of flag-XPC constructs to tethered YFP-LacR-DDB2(1–427). The values represent the percentage of colocalization of each Flag-XPC constructs with YFP on the array based on three independent experiments with SD.
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fig4: Tethering DDB2 to chromatin recruits XPC through its N- and C-terminal extremities. (A, left) Schematic representation of YFP-DDB2(1–427) and YFP-LacR-DDB2(1–427). For clarity, the sizes of the YFP (238 aa) and LacR (367 aa) were omitted. (right) Proteins from whole cell extracts (15 µg) of U2OS17 expressing either YFP-DDB2(1–427) or YFP-LacR-DDB2(1–427) were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (B–D) Recruitment of XPC (B), XPB (C), or flag-XPA (D) to tethered YFP-LacR-DDB2(1–427). The values on the graphs represent the percentage of colocalization of each NER factor with YFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (E) Schematic representation of wild-type and deleted flag-XPC constructs. (F) Recruitment of flag-XPC constructs to tethered YFP-LacR-DDB2(1–427). The values represent the percentage of colocalization of each Flag-XPC constructs with YFP on the array based on three independent experiments with SD.

Mentions: These data suggest that an immobilized NER factor can recruit the immediate downstream factors but not those upstream. To extend this observation, we introduced the LacR cassette into the YFP-DDB2(1–427) construct (Alekseev et al., 2008; Fig. 4 A). The YFP-LacR-DDB2(1–427) functionality was shown in locally irradiated cells (Fig. S1 D). As previously observed, tethering DDB2 to chromatin in U2OS17 cells caused an unfolding of the array visualized by larger spots, characteristic of decondensed chromatin (Luijsterburg et al., 2012). In agreement with our hypothesis, tethered YFP-LacR-DDB2 recruited XPC (Fig. 4 B) but not XPB and XPA (Fig. 4, C and D). These results demonstrate that PInC formation on undamaged chromatin is ordered.


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 DDB2 to chromatin recruits XPC through its N- and C-terminal extremities. (A, left) Schematic representation of YFP-DDB2(1–427) and YFP-LacR-DDB2(1–427). For clarity, the sizes of the YFP (238 aa) and LacR (367 aa) were omitted. (right) Proteins from whole cell extracts (15 µg) of U2OS17 expressing either YFP-DDB2(1–427) or YFP-LacR-DDB2(1–427) were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (B–D) Recruitment of XPC (B), XPB (C), or flag-XPA (D) to tethered YFP-LacR-DDB2(1–427). The values on the graphs represent the percentage of colocalization of each NER factor with YFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (E) Schematic representation of wild-type and deleted flag-XPC constructs. (F) Recruitment of flag-XPC constructs to tethered YFP-LacR-DDB2(1–427). The values represent the percentage of colocalization of each Flag-XPC constructs with YFP on the array based on three independent experiments with SD.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig4: Tethering DDB2 to chromatin recruits XPC through its N- and C-terminal extremities. (A, left) Schematic representation of YFP-DDB2(1–427) and YFP-LacR-DDB2(1–427). For clarity, the sizes of the YFP (238 aa) and LacR (367 aa) were omitted. (right) Proteins from whole cell extracts (15 µg) of U2OS17 expressing either YFP-DDB2(1–427) or YFP-LacR-DDB2(1–427) were resolved by SDS-PAGE followed by Western blotting using anti-GFP antibodies. (B–D) Recruitment of XPC (B), XPB (C), or flag-XPA (D) to tethered YFP-LacR-DDB2(1–427). The values on the graphs represent the percentage of colocalization of each NER factor with YFP on the array based on three independent experiments with SD. Circles indicate LacO arrays. (E) Schematic representation of wild-type and deleted flag-XPC constructs. (F) Recruitment of flag-XPC constructs to tethered YFP-LacR-DDB2(1–427). The values represent the percentage of colocalization of each Flag-XPC constructs with YFP on the array based on three independent experiments with SD.
Mentions: These data suggest that an immobilized NER factor can recruit the immediate downstream factors but not those upstream. To extend this observation, we introduced the LacR cassette into the YFP-DDB2(1–427) construct (Alekseev et al., 2008; Fig. 4 A). The YFP-LacR-DDB2(1–427) functionality was shown in locally irradiated cells (Fig. S1 D). As previously observed, tethering DDB2 to chromatin in U2OS17 cells caused an unfolding of the array visualized by larger spots, characteristic of decondensed chromatin (Luijsterburg et al., 2012). In agreement with our hypothesis, tethered YFP-LacR-DDB2 recruited XPC (Fig. 4 B) but not XPB and XPA (Fig. 4, C and D). These results demonstrate that PInC formation on undamaged chromatin is ordered.

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