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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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rad3 mutants display a gradient response to UV irradiation.(A) Survival curves of WT and different yeast rad3 mutants after UV-C exposure. (B) FACS profiles from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor, untreated or UV-irradiated with 40 or 100 J/m2 and released after 2 h. (C) Pulsed-field gel electrophoresis (PFGE) of DNA from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor and further released into S phase. Bands reveal chromosome VII by hybridization with a probe of the ADE5,7 locus. Nonlinear (NLC) and full-length linear (FLC) chromosomes include replication intermediates, in the well, and pre- and post-replicated chromosomes, which enter the gel, respectively. Bars represent the quantification of NLC with respect to the total of signal of each lane. The bottom part of the membrane below the FLC is not shown since no signals, as expected from broken DNA molecules, were revealed by hybridization in any lane. (D) All details as in (C) except that after G1 synchronization, cells were UV-irradiated with 40 J/m2 or 100 J/m2 and released into S phase 2 h later.
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pgen-1004859-g001: rad3 mutants display a gradient response to UV irradiation.(A) Survival curves of WT and different yeast rad3 mutants after UV-C exposure. (B) FACS profiles from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor, untreated or UV-irradiated with 40 or 100 J/m2 and released after 2 h. (C) Pulsed-field gel electrophoresis (PFGE) of DNA from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor and further released into S phase. Bands reveal chromosome VII by hybridization with a probe of the ADE5,7 locus. Nonlinear (NLC) and full-length linear (FLC) chromosomes include replication intermediates, in the well, and pre- and post-replicated chromosomes, which enter the gel, respectively. Bars represent the quantification of NLC with respect to the total of signal of each lane. The bottom part of the membrane below the FLC is not shown since no signals, as expected from broken DNA molecules, were revealed by hybridization in any lane. (D) All details as in (C) except that after G1 synchronization, cells were UV-irradiated with 40 J/m2 or 100 J/m2 and released into S phase 2 h later.

Mentions: To define the molecular basis of the different phenotypes of rad3-101 and rad3-107, we first studied the UV response of rad3-101 and rad3-107 mutants in comparison with that of the WT strain and the NER-deficient mutant rad3-2. Notably, rad3-101 cells respond to UV as the WT strain and in contrast to rad3-102 cells, which were slightly UV sensitive to increasing doses of UV. Instead rad3-107, as rad3-2, was highly UV-sensitive (Fig. 1A).


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)

rad3 mutants display a gradient response to UV irradiation.(A) Survival curves of WT and different yeast rad3 mutants after UV-C exposure. (B) FACS profiles from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor, untreated or UV-irradiated with 40 or 100 J/m2 and released after 2 h. (C) Pulsed-field gel electrophoresis (PFGE) of DNA from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor and further released into S phase. Bands reveal chromosome VII by hybridization with a probe of the ADE5,7 locus. Nonlinear (NLC) and full-length linear (FLC) chromosomes include replication intermediates, in the well, and pre- and post-replicated chromosomes, which enter the gel, respectively. Bars represent the quantification of NLC with respect to the total of signal of each lane. The bottom part of the membrane below the FLC is not shown since no signals, as expected from broken DNA molecules, were revealed by hybridization in any lane. (D) All details as in (C) except that after G1 synchronization, cells were UV-irradiated with 40 J/m2 or 100 J/m2 and released into S phase 2 h later.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004859-g001: rad3 mutants display a gradient response to UV irradiation.(A) Survival curves of WT and different yeast rad3 mutants after UV-C exposure. (B) FACS profiles from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor, untreated or UV-irradiated with 40 or 100 J/m2 and released after 2 h. (C) Pulsed-field gel electrophoresis (PFGE) of DNA from WT, rad3-101, rad3-107 and rad3-102 cells synchronized in G1 with α-factor and further released into S phase. Bands reveal chromosome VII by hybridization with a probe of the ADE5,7 locus. Nonlinear (NLC) and full-length linear (FLC) chromosomes include replication intermediates, in the well, and pre- and post-replicated chromosomes, which enter the gel, respectively. Bars represent the quantification of NLC with respect to the total of signal of each lane. The bottom part of the membrane below the FLC is not shown since no signals, as expected from broken DNA molecules, were revealed by hybridization in any lane. (D) All details as in (C) except that after G1 synchronization, cells were UV-irradiated with 40 J/m2 or 100 J/m2 and released into S phase 2 h later.
Mentions: To define the molecular basis of the different phenotypes of rad3-101 and rad3-107, we first studied the UV response of rad3-101 and rad3-107 mutants in comparison with that of the WT strain and the NER-deficient mutant rad3-2. Notably, rad3-101 cells respond to UV as the WT strain and in contrast to rad3-102 cells, which were slightly UV sensitive to increasing doses of UV. Instead rad3-107, as rad3-2, was highly UV-sensitive (Fig. 1A).

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