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The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

Herrera-Moyano E, Moriel-Carretero M, Montelone BA, Aguilera A - PLoS Genet. (2014)

Bottom Line: We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them.Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient.We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Seville, Spain.

ABSTRACT
The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.

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In vivo TFIIH complex dynamics in rad3 mutants.(A) FRAP analysis of asynchronous non-irradiated cultures expressing yEGFP-Tfb4. Pictures show Tfb4-yEGFP fluorescence in the nucleus of WT cells. The white square indicates the bleached area. Curves show the evolution of the normalized fluorescence in the bleached area of the nucleus [fluorescence intensity at each time-point (I) between the initial value of fluorescence intensity (Io)]. Each represented value corresponds to the median value calculated from four consecutive time-points. Error bars indicate the SD of two independent experiments. (B) FRAP analysis of asynchronous 80 J/m2 UV-C-irradiated cultures expressing yEGFP-Tfb4. Details as in (A). (C) Total protein extracts of G1-synchronized cells that were either UV-irradiated (+) or not (-) with 100 J/m2 were probed for Rad53 phosphorylation. Control and Rad3 ATP-binding groove mutant cells are compared. Unspecific hybridization of the antibody is used as an inner loading control.
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pgen-1004859-g004: In vivo TFIIH complex dynamics in rad3 mutants.(A) FRAP analysis of asynchronous non-irradiated cultures expressing yEGFP-Tfb4. Pictures show Tfb4-yEGFP fluorescence in the nucleus of WT cells. The white square indicates the bleached area. Curves show the evolution of the normalized fluorescence in the bleached area of the nucleus [fluorescence intensity at each time-point (I) between the initial value of fluorescence intensity (Io)]. Each represented value corresponds to the median value calculated from four consecutive time-points. Error bars indicate the SD of two independent experiments. (B) FRAP analysis of asynchronous 80 J/m2 UV-C-irradiated cultures expressing yEGFP-Tfb4. Details as in (A). (C) Total protein extracts of G1-synchronized cells that were either UV-irradiated (+) or not (-) with 100 J/m2 were probed for Rad53 phosphorylation. Control and Rad3 ATP-binding groove mutant cells are compared. Unspecific hybridization of the antibody is used as an inner loading control.

Mentions: We had established the affinity for DNA in vivo of the mutant Rad3 proteins by studying the residence of TFIIH at promoters. We then assayed globally the capacity of mutant TFIIH to bind to DNA at NER sites in vivo by using Fluorescence Recovery After Photo-bleaching (FRAP) using the tagged Tfb4-yEGFP protein. The rationale behind was that an impairment in the ATPase activity would lead to helicase activity defects and consequently to a defective performance of the protein during the repair reaction. Therefore, after UV, if rad3 mutations prevented these activities of the protein, a bigger diffusing fraction should be observed for the mutants. We first confirmed that Tfb4-yEGFP behaves as a wild-type non-tagged Tfb4 by showing that the EGFP signal was detected all over the nuclei, that cells were as UV-resistant as WT cells and that an expected kinetics of fluorescence recovery was observed after photo-bleaching (Fig. 4A, and S5A and S5B Figure). In untreated WT cells, full recovery of fluorescence was observed in less than 12 seconds after bleaching (Fig. 4A and S5C Figure), consistent with the observation that most of the TFIIH complex within the nucleus is diffusible [22]. After UV irradiation a variable fraction of TFIIH is expected to move to the sites of DNA lesions to be engaged in their repair, leading to a low-diffusible fraction that reduces fluorescence recovery after bleaching. Accordingly, fluorescence recovery reached only up to 80% in cells treated with an 80 J/m2 UV dose (S5C Figure).


The rem mutations in the ATP-binding groove of the Rad3/XPD helicase lead to Xeroderma pigmentosum-Cockayne syndrome-like phenotypes.

Herrera-Moyano E, Moriel-Carretero M, Montelone BA, Aguilera A - PLoS Genet. (2014)

In vivo TFIIH complex dynamics in rad3 mutants.(A) FRAP analysis of asynchronous non-irradiated cultures expressing yEGFP-Tfb4. Pictures show Tfb4-yEGFP fluorescence in the nucleus of WT cells. The white square indicates the bleached area. Curves show the evolution of the normalized fluorescence in the bleached area of the nucleus [fluorescence intensity at each time-point (I) between the initial value of fluorescence intensity (Io)]. Each represented value corresponds to the median value calculated from four consecutive time-points. Error bars indicate the SD of two independent experiments. (B) FRAP analysis of asynchronous 80 J/m2 UV-C-irradiated cultures expressing yEGFP-Tfb4. Details as in (A). (C) Total protein extracts of G1-synchronized cells that were either UV-irradiated (+) or not (-) with 100 J/m2 were probed for Rad53 phosphorylation. Control and Rad3 ATP-binding groove mutant cells are compared. Unspecific hybridization of the antibody is used as an inner loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263401&req=5

pgen-1004859-g004: In vivo TFIIH complex dynamics in rad3 mutants.(A) FRAP analysis of asynchronous non-irradiated cultures expressing yEGFP-Tfb4. Pictures show Tfb4-yEGFP fluorescence in the nucleus of WT cells. The white square indicates the bleached area. Curves show the evolution of the normalized fluorescence in the bleached area of the nucleus [fluorescence intensity at each time-point (I) between the initial value of fluorescence intensity (Io)]. Each represented value corresponds to the median value calculated from four consecutive time-points. Error bars indicate the SD of two independent experiments. (B) FRAP analysis of asynchronous 80 J/m2 UV-C-irradiated cultures expressing yEGFP-Tfb4. Details as in (A). (C) Total protein extracts of G1-synchronized cells that were either UV-irradiated (+) or not (-) with 100 J/m2 were probed for Rad53 phosphorylation. Control and Rad3 ATP-binding groove mutant cells are compared. Unspecific hybridization of the antibody is used as an inner loading control.
Mentions: We had established the affinity for DNA in vivo of the mutant Rad3 proteins by studying the residence of TFIIH at promoters. We then assayed globally the capacity of mutant TFIIH to bind to DNA at NER sites in vivo by using Fluorescence Recovery After Photo-bleaching (FRAP) using the tagged Tfb4-yEGFP protein. The rationale behind was that an impairment in the ATPase activity would lead to helicase activity defects and consequently to a defective performance of the protein during the repair reaction. Therefore, after UV, if rad3 mutations prevented these activities of the protein, a bigger diffusing fraction should be observed for the mutants. We first confirmed that Tfb4-yEGFP behaves as a wild-type non-tagged Tfb4 by showing that the EGFP signal was detected all over the nuclei, that cells were as UV-resistant as WT cells and that an expected kinetics of fluorescence recovery was observed after photo-bleaching (Fig. 4A, and S5A and S5B Figure). In untreated WT cells, full recovery of fluorescence was observed in less than 12 seconds after bleaching (Fig. 4A and S5C Figure), consistent with the observation that most of the TFIIH complex within the nucleus is diffusible [22]. After UV irradiation a variable fraction of TFIIH is expected to move to the sites of DNA lesions to be engaged in their repair, leading to a low-diffusible fraction that reduces fluorescence recovery after bleaching. Accordingly, fluorescence recovery reached only up to 80% in cells treated with an 80 J/m2 UV dose (S5C Figure).

Bottom Line: We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them.Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient.We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters.

View Article: PubMed Central - PubMed

Affiliation: Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Seville, Spain.

ABSTRACT
The eukaryotic TFIIH complex is involved in Nucleotide Excision Repair and transcription initiation. We analyzed three yeast mutations of the Rad3/XPD helicase of TFIIH known as rem (recombination and mutation phenotypes). We found that, in these mutants, incomplete NER reactions lead to replication fork breaking and the subsequent engagement of the homologous recombination machinery to restore them. Nevertheless, the penetrance varies among mutants, giving rise to a phenotype gradient. Interestingly, the mutations analyzed reside at the ATP-binding groove of Rad3 and in vivo experiments reveal a gain of DNA affinity upon damage of the mutant Rad3 proteins. Since mutations at the ATP-binding groove of XPD in humans are present in the Xeroderma pigmentosum-Cockayne Syndrome (XP-CS), we recreated rem mutations in human cells, and found that these are XP-CS-like. We propose that the balance between the loss of helicase activity and the gain of DNA affinity controls the capacity of TFIIH to open DNA during NER, and its persistence at both DNA lesions and promoters. This conditions NER efficiency and transcription resumption after damage, which in human cells would explain the XP-CS phenotype, opening new perspectives to understand the molecular basis of the role of XPD in human disease.

Show MeSH
Related in: MedlinePlus