Cockayne syndrome, between transcription and DNA repair defects.
Explore the source record for details and available documents.
Cockayne Syndrome↗
Biomedical subjects
J M Egly
Publications and source records attributed to J M Egly.
TFIIH functions are altered by the P210BCR-ABL oncoprotein produced on the Philadelphia chromosome.
P210BCR-ABL counteracted against the complementary effect of XPB on DNA repair when ultraviolet (UV)-sensitive 27-1 cells were treated with UV or cisplatin but not with hydrogen peroxide. Wortmannin, an inhibitor of PI3 kinase did not affect its anti-repair effect. Enhanced recruitment of p44 with TFIIH after cisplatin treatment is inhibited by the expression of P210BCR-ABL in a kinase activity-dependent manner. Although purified TFIIH from P210BCR-ABL expressor and non-expressor showed almost no difference in molar ratio of each component, the in vitro activity of TFIIH was decreased by 5-10% in repair assay but was increased by more than two-fold in transcription assay.
Animals↗
TFIIH inhibits CDK9 phosphorylation during human immunodeficiency virus type 1 transcription.
Tat stimulates human immunodeficiency virus, type 1 (HIV-1), transcription elongation by recruitment of the human transcription elongation factor P-TEFb, consisting of CDK9 and cyclin T1, to the TAR RNA structure. It has been demonstrated further that CDK9 phosphorylation is required for high affinity binding of Tat/P-TEFb to the TAR RNA structure and that the state of P-TEFb phosphorylation may regulate Tat transactivation. We now demonstrate that CDK9 phosphorylation is uniquely regulated in the HIV-1 preinitiation and elongation complexes. The presence of TFIIH in the HIV-1 preinitiation complex inhibits CDK9 phosphorylation. As TFIIH is released from the elongation complex between +14 and +36, CDK9 phosphorylation is observed. In contrast to the activity in the "soluble" complex, phosphorylation of CDK9 is increased by the presence of Tat in the transcription complexes. Consistent with these observations, we have demonstrated that purified TFIIH directly inhibits CDK9 autophosphorylation. By using recombinant TFIIH subcomplexes, our results suggest that the XPB subunit of TFIIH is responsible for this inhibition of CDK9 phosphorylation. Interestingly, our results further suggest that the phosphorylated form of CDK9 is the active kinase for RNA polymerase II carboxyl-terminal domain phosphorylation.
Blotting, Western↗
A yeast four-hybrid system identifies Cdk-activating kinase as a regulator of the XPD helicase, a subunit of transcription factor IIH.
To understand the role of the various components of TFIIH, a DNA repair/transcription factor, a yeast four-hybrid system was designed. When the ternary Cdk-activating kinase (CAK) complex composed of Cdk7, cyclin H, and MAT1 was used as bait, the xeroderma pigmentosum (XP) D helicase of transcription factor IIH (TFIIH), among other proteins, was identified as an interacting partner. Deletion mutant analyses demonstrated that the coiled-coil and the hydrophobic domains of MAT1 interlink the CAK complex directly with the N-terminal domain of XPD. Using immunoprecipitates from cells coinfected with baculoviruses, we further validated the bridging function of XPD, which anchors CAK to the core TFIIH. In addition we show that upon interaction with MAT1, CAK inhibits the helicase activity of XPD. This inhibition is overcome upon binding to p44, a subunit of the core TFIIH. It is not surprising that under these conditions some XPD mutations affect interactions not only with p44, but also with MAT1, thus preventing either the CAK inhibitory function within CAK.XPD and/or the role of CAK within TFIIH and, consequently, explaining the variety of the XP phenotypes.
Base Sequence↗
The 14th Datta Lecture. TFIIH: from transcription to clinic.
Once a large proportion of the genes responsible for genetic disorders are identified in the post-genome era, the fundamental challenge is to establish a genotype/phenotype relationship. Our aim is to explain how mutations in a given gene affect its enzymatic function and, in consequence, disturb the life of the cell. Genome integrity is continuously threatened by the occurrence of DNA damage arising from cellular exposure to irradiation and genotoxic chemicals. This mutagenic or potentially lethal DNA damage induces various cellular responses including cell cycle arrest, transcription alteration and processing by DNA repair mechanisms, such as the nucleotide excision repair (NER) pathway. Disruption of NER in response to genotoxic injuries results in autosomal recessive hereditary diseases such as Xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). One of the most immediate consequences of the induction of strand-distorting lesions is the arrest of transcription in which TFIIH plays a role in addition to its role in DNA repair. The observations made by clinicians close to XP, TTD and CS patients, suggested that transcription defects responsible for brittle hair and nails for TTD, or developmental abnormalities for CS, resulted from TFIIH mutations. Here a story will be related which could be called 'a multi-faceted factor named TFIIH'. As biochemists, we have characterized each component of TFIIH, three of which are XPB and XPD helicases and cdk7, a cyclin-dependent kinase. With the help of structural biologists, we have characterized most of the specific three-dimensional structures of TFIIH subunits and obtained its electron microscopy image. Together these approaches help us to propose a number of structure-function relationships for TFIIH. Through transfection and microinjection assays, cell biology allows us to determine the role of TFIIH in transcription and NER. We are thus in a position to explain, at least in part, transcription initiation mechanisms and their coupling to DNA repair. We now know how the XPB helicase opens the promoter region for RNA synthesis and that one of the roles of XPD helicase is to anchor the cdk7 kinase to the core-TFIIH. In XP and CS associated patients, we have demonstrated that some XPD mutations prevent an optimal phosphorylation of nuclear receptors by cdk7 with, as a consequence, a drop in the expression of genes sensitive to hormone action. We have thus shown that hormonal responses operate through TFIIH. Careful analysis of each TFIIH subunit also shows how the p44 Ring finger participates in certain promoter escape reactions. We are also able to localize the action of TFIIH in the sequence of events that lead to the elimination of DNA lesions. Thanks to the combination of these different approaches we are obtaining a much clearer picture of the TFIIH complex and its integration into the life of the cell.
Cell Cycle↗
A role of the C-terminal part of p44 in the promoter escape activity of transcription factor IIH.
The p44 subunit plays a crucial role in the overall activity of the transcription/DNA repair factor TFIIH: on the one hand its N-terminal domain interacts with and regulates the XPD helicase (, ); on the other hand, as shown in the present study, it participates with the promoter escape reaction. Mutagenesis along with recombinant technology using the baculovirus/insect cells expression system allowed us to define the function of the two structural motifs of the C-terminal moiety of p44: mutations within the C4 zinc finger motif (residues 291-308) prevent incorporation of the p62 subunit within the core TFIIH. Double mutations in the RING finger motif (residues 345-385) allow the synthesis of the first phosphodiester bond by RNA polymerase II, but prevent its escape from the promoter. This highlights the role of transcription factor IIH in the various steps of the transcription initiation process.
Animals↗
Codominance associated with overexpression of certain XPD mutations.
Mutations in the XPD gene are associated with three complex clinical phenotypes, namely xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (XP-CS), and trichothiodystrophy (TTD). XP is caused by a deficiency in nucleotide excision repair (NER) that results in a high risk of skin cancer. TTD is characterized by severe developmental and neurological defects, with hallmark features of brittle hair and scaly skin, and sometimes has defective NER. We used CHO cells as a system to study how specific mutations alter the dominant/recessive behavior of XPD protein. Previously we identified the T46I and R75W mutations in two highly UV-sensitive hamster cell lines that were reported to have paradoxically high levels of unscheduled DNA synthesis. Here we report that these mutants have greatly reduced XPD helicase activity and fully defective NER in a cell-extract excision assay. We conclude that the unscheduled DNA synthesis seen in these mutants is caused by abortive "repair" that does not contribute to cell survival. These mutations, as well as the K48R canonical helicase-domain mutation, each produced codominant negative phenotypes when overexpressed in wild-type CHO cells. The common XP-specific R683W mutation also behaved in a codominant manner when overexpressed, which is consistent with the idea that this mutation may affect primarily the enzymatic activity of the protein rather than impairing protein interactions, which may underlie TTD. A C-terminal mutation uniquely found in TTD (R722W) was overexpressed but not to levels sufficiently high to rigorously test for a codominant phenotype. Overexpression of mutant XPD alleles may provide a simple means of producing NER deficiency in other cell lines.
Animals↗
Trichothiodystrophy, a transcription syndrome.
Trichothiodystrophy (TTD) is a rare genetic disorder characterized by a hair dysplasia and associated with numerous symptoms affecting mainly organs derived from the neuroectoderm. About half of TTD patients exhibit photosensitivity because their nucleotide-excision repair pathway (NER) does not remove UV-induced DNA lesions efficiently. However, they do not present the skin cancer susceptibility expected from such an NER disorder. Their deficiencies result from phenotype-specific mutations in either XPB or XPD. These genes encode the helicase subunits of TFIIH, a DNA repair factor that is also required for transcription of class II genes. Thus, time- and tissue-specific impairments of transcription might explain the developmental and neurological symptoms of TTD. In a third group of photosensitive patients, TTD-A, no mutation has been identified, although TFIIH amount is reduced.
Abnormalities, Multiple↗
Three-hybrid screens. Inducible third-party systems.
Explore the source record for details and available documents.
Culture Media↗
Cloning of a human homolog of the yeast nucleotide excision repair gene MMS19 and interaction with transcription repair factor TFIIH via the XPB and XPD helicases.
Nucleotide excision repair (NER) removes UV-induced photoproducts and numerous other DNA lesions in a highly conserved 'cut-and-paste' reaction that involves approximately 25 core components. In addition, several other proteins have been identified which are dispensable for NER in vitro but have an undefined role in vivo and may act at the interface of NER and other cellular processes. An intriguing example is the Saccharomyces cerevisiae Mms19 protein that has an unknown dual function in NER and RNA polymerase II transcription. Here we report the cloning and characterization of a human homolog, designated hMMS19, that encodes a 1030 amino acid protein with 26% identity and 51% similarity to S.cerevisiae Mms19p and with a strikingly similar size. The expression profile and nuclear location are consistent with a repair function. Co-immunoprecipitation experiments revealed that hMMS19 directly interacts with the XPB and XPD subunits of NER-transcription factor TFIIH. These findings extend the conservation of the NER apparatus and the link between NER and basal transcription and suggest that hMMS19 exerts its function in repair and transcription by interacting with the XPB and XPD helicases.
Amino Acid Sequence↗
Solution structure of the N-terminal domain of the human TFIIH MAT1 subunit: new insights into the RING finger family.
The human MAT1 protein belongs to the cyclin-dependent kinase-activating kinase complex, which is functionally associated to the transcription/DNA repair factor TFIIH. The N-terminal region of MAT1 consists of a C3HC4 RING finger, which contributes to optimal TFIIH transcriptional activities. We report here the solution structure of the human MAT1 RING finger domain (Met(1)-Asp(65)) as determined by (1)H NMR spectroscopy. The MAT1 RING finger domain presents the expected betaalphabetabeta topology with two interleaved zinc-binding sites conserved among the RING family. However, the presence of an additional helical segment in the N-terminal part of the domain and a conserved hydrophobic central beta strand are the defining features of this new structure and more generally of the MAT1 RING finger subfamily. Comparison of electrostatic surfaces of RING finger structures shows that the RING finger domain of MAT1 presents a remarkable positively charged surface. The functional implications of these MAT1 RING finger features are discussed.
Amino Acid Sequence↗
p44/SSL1, the regulatory subunit of the XPD/RAD3 helicase, plays a crucial role in the transcriptional activity of TFIIH.
In order to unravel the mechanism that regulates transcription of protein-coding genes, we investigated the function of the p44 subunit of TFIIH, a basal transcription factor that is also involved in DNA repair. We have shown previously that mutations in the C terminus of the XPD helicase, another subunit of TFIIH, prevent its regulation by p44 (Coin, F., Bergmann, E., Tremeau-Bravard, A., and Egly, J. M. (1999) EMBO 18, 1357-1366). By using a site-directed mutagenesis approach within the p44 region from amino acids 66 to 200, we indicate how a decrease in the interaction between p44 and XPD results in a decrease of the XPD helicase activity and leads to a defect in the first steps of the transcription reaction, namely the first phosphodiester bond formation and promoter clearance. We thus provide some explanation for the transcriptional defect found in SSL1 mutated yeast (Wang, Z., Buratowski, S., Svejstrup, J. Q., Feaver, W. J., Wu, X., Kornberg, R. D., Donahue, T. F., and Friedberg, E. C. (1995) Mol. Cell. Biol. 15, 2288-2293). Moreover, this study shows how the activity of the the cyclin-dependent kinase-activating kinase associated with TFIIH complex in stimulating transcription is mediated in part by p44/XPD interaction.
Adenosine Triphosphatases↗
Structural characterization of the cysteine-rich domain of TFIIH p44 subunit.
In an effort to understand the structure function relationship of TFIIH, a transcription/repair factor, we focused our attention on the p44 subunit, which plays a central role in both mechanisms. The amino-terminal portion of p44 has been shown to be involved in the regulation of the XPD helicase activity; here we show that its carboxyl-terminal domain is essential for TFIIH transcription activity and that it binds three zinc atoms through two independent modules. The first contains a C4 zinc finger motif, whereas the second is characterized by a CX(2)CX(2-4)FCADCD motif, corresponding to interleaved zinc binding sites. The solution structure of this second module reveals an unexpected homology with the regulatory domain of protein kinase C and provides a framework to study its role at the molecular level.
Amino Acid Sequence↗
Molecular structure of human TFIIH.
TFIIH is a multiprotein complex required for both transcription and DNA repair. Single particles of human TFIIH were revealed by electron microscopy and image processing at a resolution of 3.8 nm. TFIIH is 16 x 12.5 x 7.5 nm in size and is organized into a ring-like structure from which a large protein domain protrudes out. A subcomplex assembled from five recombinant core subunits also forms a circular architecture that can be superimposed on the ring found in human TFIIH. Immunolabeling experiments localize several subunits: p44, within the ring structure, forms the base of the protruding protein density which includes the cdk7 kinase, cyclin H, and MAT1. Within the ring structure, p44 was flanked on either side by the XPB and XPD helicases. These observations provide us with a quartenary organizational model of TFIIH.
Antibodies, Monoclonal↗
Distinct regions of MAT1 regulate cdk7 kinase and TFIIH transcription activities.
The transcription/DNA repair factor TFIIH may be resolved into at least two subcomplexes: the core TFIIH and the cdk-activating kinase (CAK) complex. The CAK complex, which is also found free in the cell, is composed of cdk7, cyclin H, and MAT1. In the present work, we found that the C terminus of MAT1 binds to the cdk7 x cyclin H complex and activates the cdk7 kinase activity. The median portion of MAT1, which contains a coiled-coil motif, allows the binding of CAK to the TFIIH core through interactions with both XPD and XPB helicases. Furthermore, using recombinant TFIIH complexes, it is demonstrated that the N-terminal RING finger domain of MAT1 is crucial for transcription activation and participates to the phosphorylation of the C-terminal domain of the largest subunit of the RNA polymerase II.
Amino Acid Sequence↗
TFIIH interacts with the retinoic acid receptor gamma and phosphorylates its AF-1-activating domain through cdk7.
Retinoic acid receptor gamma (RARgamma) is phosphorylated in COS-1 cells at two conserved serine residues located in the N-terminal region (serines 77 and 79 in RARgamma1 and serines 66 and 68 in RARgamma2) that contains the activation function AF-1. These serines are phosphorylated in vitro by cdk7, a cyclin-dependent kinase associated to cyclin H and MAT1 in the CAK complex (cdk7.cyclin H. MAT1), that is found either free or as a component of the transcription/DNA repair factor TFIIH. RARgamma is more efficiently phosphorylated by TFIIH than by CAK and interacts not only with cdk7 but also with several additional subunits of TFIIH. RARgamma phosphorylation and interaction with TFIIH occur in a ligand-independent manner. Our data demonstrate also that phosphorylation of the AF-1 function modulates RARgamma transcriptional activity in a response gene-dependent manner.
Animals↗
TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair.
TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5' nor 3' incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients.
Adenosine Triphosphatases↗
Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK.
During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase delta or epsilon, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.
Cyclin-Dependent Kinases↗