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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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Analysis of TFIIH recruitment to promoters in rad3 mutants.(A) Chromatin Immunoprecipitation (ChIP) analysis of Tfb4-TAP. Cells were grown in synthetic complete (SC) medium until the exponential phase. ChIP analysis was performed at the ALG9 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. (B) ChIP analysis of Tfb4-TAP after UV damage. Cells were grown in SC medium until the exponential phase, and then irradiated with 80 J/m2. Analysis of the different time-point samples was performed at the GRX1 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. The mean and the SD of triplicate assays of four independent experiments are depicted for each condition. *, p<0.05, **, p<0.01 (Student's t-test).
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pgen-1004859-g003: Analysis of TFIIH recruitment to promoters in rad3 mutants.(A) Chromatin Immunoprecipitation (ChIP) analysis of Tfb4-TAP. Cells were grown in synthetic complete (SC) medium until the exponential phase. ChIP analysis was performed at the ALG9 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. (B) ChIP analysis of Tfb4-TAP after UV damage. Cells were grown in SC medium until the exponential phase, and then irradiated with 80 J/m2. Analysis of the different time-point samples was performed at the GRX1 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. The mean and the SD of triplicate assays of four independent experiments are depicted for each condition. *, p<0.05, **, p<0.01 (Student's t-test).

Mentions: First, we asked whether mutations in the ATP-binding groove of Rad3, such as those of the rem strains studied here, could cause a gain in DNA affinity in vivo, independently of whether or not being masked by a helicase activity defect. For this we analyzed TFIIH retention at promoters, in which the helicase activity needed to open the DNA is provided by Rad25 and not by Rad3 [5]. We performed Tfb4-TAP chromatin immunoprecipitation (ChIP) in asynchronous cultures of the wild-type strain, the three rem mutants and the NER- mutant rad3-2, plus the rad3-25 mutant carrying a E548K amino acid change that maps at the DNA binding channel (S4A Figure), outside of the ATP-binding groove and that was therefore expected not to show a significant gain of ssDNA affinity. To minimize any possible effect caused by transcription, we determined TFIIH binding at the ALG9 promoter, since ALG9 is constitutively expressed at a constant rate independently of environmental and cellular conditions [20] and we verified that it was transcribed in all mutants to similar levels as the WT (S4B Figure). The ChIP analyses show that TFIIH is more abundant at the ALG9 promoter in the three rem mutants, while in the rad3-25 control recruitment was indistinguishable from the WT (Fig. 3A). The rad3-2 NER- mutant displayed the strongest promoter retention of all ATP-groove mutants, up to 3-fold the WT levels (Fig. 3A).


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)

Analysis of TFIIH recruitment to promoters in rad3 mutants.(A) Chromatin Immunoprecipitation (ChIP) analysis of Tfb4-TAP. Cells were grown in synthetic complete (SC) medium until the exponential phase. ChIP analysis was performed at the ALG9 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. (B) ChIP analysis of Tfb4-TAP after UV damage. Cells were grown in SC medium until the exponential phase, and then irradiated with 80 J/m2. Analysis of the different time-point samples was performed at the GRX1 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. The mean and the SD of triplicate assays of four independent experiments are depicted for each condition. *, p<0.05, **, p<0.01 (Student's t-test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263401&req=5

pgen-1004859-g003: Analysis of TFIIH recruitment to promoters in rad3 mutants.(A) Chromatin Immunoprecipitation (ChIP) analysis of Tfb4-TAP. Cells were grown in synthetic complete (SC) medium until the exponential phase. ChIP analysis was performed at the ALG9 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. (B) ChIP analysis of Tfb4-TAP after UV damage. Cells were grown in SC medium until the exponential phase, and then irradiated with 80 J/m2. Analysis of the different time-point samples was performed at the GRX1 promoter and normalized with respect to the MFA2 promoter in MATα cells, which is constitutively repressed. The mean and the SD of triplicate assays of four independent experiments are depicted for each condition. *, p<0.05, **, p<0.01 (Student's t-test).
Mentions: First, we asked whether mutations in the ATP-binding groove of Rad3, such as those of the rem strains studied here, could cause a gain in DNA affinity in vivo, independently of whether or not being masked by a helicase activity defect. For this we analyzed TFIIH retention at promoters, in which the helicase activity needed to open the DNA is provided by Rad25 and not by Rad3 [5]. We performed Tfb4-TAP chromatin immunoprecipitation (ChIP) in asynchronous cultures of the wild-type strain, the three rem mutants and the NER- mutant rad3-2, plus the rad3-25 mutant carrying a E548K amino acid change that maps at the DNA binding channel (S4A Figure), outside of the ATP-binding groove and that was therefore expected not to show a significant gain of ssDNA affinity. To minimize any possible effect caused by transcription, we determined TFIIH binding at the ALG9 promoter, since ALG9 is constitutively expressed at a constant rate independently of environmental and cellular conditions [20] and we verified that it was transcribed in all mutants to similar levels as the WT (S4B Figure). The ChIP analyses show that TFIIH is more abundant at the ALG9 promoter in the three rem mutants, while in the rad3-25 control recruitment was indistinguishable from the WT (Fig. 3A). The rad3-2 NER- mutant displayed the strongest promoter retention of all ATP-groove mutants, up to 3-fold the WT levels (Fig. 3A).

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