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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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Evidences of DNA damage in XPD-102 human cells.(A) Immunofluorescence of U2OS cells and quantification of the number of U2OS and XP16BR cells containing 53BP1 foci after transfection with pIRES2-EGFP empty vector (Control), pIRES2-EGFP-XPD overexpressing the wild-type XPD allele (XPD) and pIRES2-EGFP-XPD-102 overexpressing the XPD-102 rem allele (XPD-102). Expression of EGFP is used as a marker of transfection. Error bars indicate SD of three independent experiments. (B) Density map of the DNA single-cell electrophoresis from U2OS cells and quantification of the comet tail moments of U2OS and XP16BR cells transfected with the control, XPD and XPD-102 constructions. Details as in (A) (C) Immunofluorescence and quantification data of the number of U2OS Control, XPD and XPD-102 cells containing more than 3 γH2AX foci after transfection with siControl (siC) or siXPA. Error bars indicate the SD of four independent experiments. (D) Immunofluorescence and quantification of the number of U2OS control, XPD and XPD-102 cells containing either pan-nuclear staining or more than 10 discrete γH2AX foci 5 hours after irradiation with 5 J/m2 UV-C. Error bars indicate the SD of four independent experiments. *, p<0.05 (Mann-Whitney U test).
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pgen-1004859-g005: Evidences of DNA damage in XPD-102 human cells.(A) Immunofluorescence of U2OS cells and quantification of the number of U2OS and XP16BR cells containing 53BP1 foci after transfection with pIRES2-EGFP empty vector (Control), pIRES2-EGFP-XPD overexpressing the wild-type XPD allele (XPD) and pIRES2-EGFP-XPD-102 overexpressing the XPD-102 rem allele (XPD-102). Expression of EGFP is used as a marker of transfection. Error bars indicate SD of three independent experiments. (B) Density map of the DNA single-cell electrophoresis from U2OS cells and quantification of the comet tail moments of U2OS and XP16BR cells transfected with the control, XPD and XPD-102 constructions. Details as in (A) (C) Immunofluorescence and quantification data of the number of U2OS Control, XPD and XPD-102 cells containing more than 3 γH2AX foci after transfection with siControl (siC) or siXPA. Error bars indicate the SD of four independent experiments. (D) Immunofluorescence and quantification of the number of U2OS control, XPD and XPD-102 cells containing either pan-nuclear staining or more than 10 discrete γH2AX foci 5 hours after irradiation with 5 J/m2 UV-C. Error bars indicate the SD of four independent experiments. *, p<0.05 (Mann-Whitney U test).

Mentions: In the second place, we wanted to test whether the rem alleles had the same impact on human cells as in S. cerevisiae. For this, we first assayed whether the yeast rad3-102 feature of accumulation of spontaneous DNA breaks was recreated in human cells. To achieve it, we overexpressed an XPD allele, XPD-102 (XPD-H659Y), carrying the equivalent of the yeast semi-dominant rad3-102 (rad3-H661Y) mutation (Fig. 5A). As a control we overexpressed the wild-type version of XPD from the same plasmid. mRNA levels of XPD and XPD-102 increased 300-fold with respect to cells transfected with the empty vector after 24 hours of transfection, and this correlated with increased expression at the protein level (S6A Figure). As an additional control, we verified that XPD-102 overexpression did not alter basic transcriptional patterns of the cell. Analysis by qPCR of levels of two relevant housekeeping mRNAs denoted no change in transcription between control and XPD-102-overexpressing cells (S6B 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)

Evidences of DNA damage in XPD-102 human cells.(A) Immunofluorescence of U2OS cells and quantification of the number of U2OS and XP16BR cells containing 53BP1 foci after transfection with pIRES2-EGFP empty vector (Control), pIRES2-EGFP-XPD overexpressing the wild-type XPD allele (XPD) and pIRES2-EGFP-XPD-102 overexpressing the XPD-102 rem allele (XPD-102). Expression of EGFP is used as a marker of transfection. Error bars indicate SD of three independent experiments. (B) Density map of the DNA single-cell electrophoresis from U2OS cells and quantification of the comet tail moments of U2OS and XP16BR cells transfected with the control, XPD and XPD-102 constructions. Details as in (A) (C) Immunofluorescence and quantification data of the number of U2OS Control, XPD and XPD-102 cells containing more than 3 γH2AX foci after transfection with siControl (siC) or siXPA. Error bars indicate the SD of four independent experiments. (D) Immunofluorescence and quantification of the number of U2OS control, XPD and XPD-102 cells containing either pan-nuclear staining or more than 10 discrete γH2AX foci 5 hours after irradiation with 5 J/m2 UV-C. Error bars indicate the SD of four independent experiments. *, p<0.05 (Mann-Whitney U test).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4263401&req=5

pgen-1004859-g005: Evidences of DNA damage in XPD-102 human cells.(A) Immunofluorescence of U2OS cells and quantification of the number of U2OS and XP16BR cells containing 53BP1 foci after transfection with pIRES2-EGFP empty vector (Control), pIRES2-EGFP-XPD overexpressing the wild-type XPD allele (XPD) and pIRES2-EGFP-XPD-102 overexpressing the XPD-102 rem allele (XPD-102). Expression of EGFP is used as a marker of transfection. Error bars indicate SD of three independent experiments. (B) Density map of the DNA single-cell electrophoresis from U2OS cells and quantification of the comet tail moments of U2OS and XP16BR cells transfected with the control, XPD and XPD-102 constructions. Details as in (A) (C) Immunofluorescence and quantification data of the number of U2OS Control, XPD and XPD-102 cells containing more than 3 γH2AX foci after transfection with siControl (siC) or siXPA. Error bars indicate the SD of four independent experiments. (D) Immunofluorescence and quantification of the number of U2OS control, XPD and XPD-102 cells containing either pan-nuclear staining or more than 10 discrete γH2AX foci 5 hours after irradiation with 5 J/m2 UV-C. Error bars indicate the SD of four independent experiments. *, p<0.05 (Mann-Whitney U test).
Mentions: In the second place, we wanted to test whether the rem alleles had the same impact on human cells as in S. cerevisiae. For this, we first assayed whether the yeast rad3-102 feature of accumulation of spontaneous DNA breaks was recreated in human cells. To achieve it, we overexpressed an XPD allele, XPD-102 (XPD-H659Y), carrying the equivalent of the yeast semi-dominant rad3-102 (rad3-H661Y) mutation (Fig. 5A). As a control we overexpressed the wild-type version of XPD from the same plasmid. mRNA levels of XPD and XPD-102 increased 300-fold with respect to cells transfected with the empty vector after 24 hours of transfection, and this correlated with increased expression at the protein level (S6A Figure). As an additional control, we verified that XPD-102 overexpression did not alter basic transcriptional patterns of the cell. Analysis by qPCR of levels of two relevant housekeeping mRNAs denoted no change in transcription between control and XPD-102-overexpressing cells (S6B 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