Miria Stefanini

The Cockayne syndrome group A gene encodes a WD repeat protein that interacts with CSB protein and a subunit of RNA polymerase II TFIIH

The hereditary disease Cockayne syndrome (CS) is characterized by a complex clinical phenotype. CS cells are abnormally sensitive to ultraviolet radiation and are defective in the repair of transcriptionally active genes. The cloned CSB gene encodes a member of a protein family that includes the yeast Snf2 protein, a component of the transcriptional regulator Swi/Snf. We report the cloning of the CSA cDNA, which can encode a WD repeat protein. Mutations in the cDNA have been identified in CS-A cell lines. CSA protein interacts with CSB protein and with p44 protein, a subunit of the human RNA polymerase II transcription factor IIH. These observations suggest that the products of the CSA and CSB genes are involved in transcription.

A new, tenth subunit of TFIIH is responsible for the DNA repair syndrome trichothiodystrophy group A

DNA repair-deficient trichothiodystrophy (TTD) results from mutations in the XPD and XPB subunits of the DNA repair and transcription factor TFIIH. In a third form of DNA repair–deficient TTD, called group A, none of the nine subunits encoding TFIIH carried mutations; instead, the steady-state level of the entire complex was severely reduced. A new, tenth TFIIH subunit (TFB5) was recently identified in yeast. Here, we describe the identification of the human TFB5 ortholog and its association with human TFIIH. Microinjection of cDNA encoding TFB5 (GTF2H5, also called TTDA) corrected the DNA-repair defect of TTD-A cells, and we identified three functional inactivating mutations in this gene in three unrelated families with TTD-A. The GTF2H5 gene product has a role in regulating the level of TFIIH. The identification of a new evolutionarily conserved subunit of TFIIH implicated in TTD-A provides insight into TFIIH function in transcription, DNA repair and human disease.

New functions of XPC in the protection of human skin cells from oxidative damage

Xeroderma pigmentosum (XP) C is involved in the recognition of a variety of bulky DNA‐distorting lesions in nucleotide excision repair. Here, we show that XPC plays an unexpected and multifaceted role in cell protection from oxidative DNA damage. XP‐C primary keratinocytes and fibroblasts are hypersensitive to the killing effects of DNA‐oxidizing agents and this effect is reverted by expression of wild‐type XPC. Upon oxidant exposure, XP‐C primary keratinocytes and fibroblasts accumulate 8,5′‐cyclopurine 2′‐deoxynucleosides in their DNA, indicating that XPC is involved in their removal. In the absence of XPC, a decrease in the repair rate of 8‐hydroxyguanine (8‐OH‐Gua) is also observed. We demonstrate that XPC–HR23B complex acts as cofactor in base excision repair of 8‐OH‐Gua, by stimulating the activity of its specific DNA glycosylase OGG1. In vitro experiments suggest that the mechanism involved is a combination of increased loading and turnover of OGG1 by XPC‐HR23B complex. The accumulation of endogenous oxidative DNA damage might contribute to increased skin cancer risk and account for internal cancers reported for XP‐C patients.

Basal Transcription Defect Discriminates between Xeroderma Pigmentosum and Trichothiodystrophy in XPD Patients

Mutations in the XPD gene result in xeroderma pigmentosum (XP) and trichothiodystrophy (TTD), the phenotypes of which are often intricate. To understand the genotype/phenotype relationship, we engineered recombinant TFIIHs in which XPD subunits carry amino acid changes found in XPD patients. We demonstrate that all the XPD mutations are detrimental for XPD helicase activity, thus explaining the NER defect. We also show that TFIIH from TTD patients, but not from XP patients, exhibits a significant in vitro basal transcription defect in addition to a reduced intracellular concentration. Moreover, when XPD mutations prevent interaction with the p44 subunit of TFIIH, transactivation directed by certain nuclear receptors is inhibited, regardless of TTD versus XP phenotype, thus explaining the overlapping symptoms. The implications of these mutations are discussed using a structural model of the XPD protein. Our study provides explanations for the nature and the severity of the various clinical features.

Molecular Analysis of Mutations in the CSB(ERCC6) Gene in Patients with Cockayne Syndrome

Cockayne syndrome is a multisystem sun-sensitive genetic disorder associated with a specific defect in the ability to perform transcription-coupled repair of active genes after UV irradiation. Two complementation groups (CS-A and CS-B) have been identified, and 80% of patients have been assigned to the CS-B complementation group. We have analyzed the sites of the mutations in the CSB gene in 16 patients, to determine the spectrum of mutations in this gene and to see whether the nature of the mutation correlates with the type and severity of the clinical symptoms. In nine of the patients, the mutations resulted in truncated products in both alleles, whereas, in the other seven, at least one allele contained a single amino acid change. The latter mutations were confined to the C-terminal two-thirds of the protein and were shown to be inactivating by their failure to restore UV-irradiation resistance to hamster UV61 cells, which are known to be defective in the CSB gene. Neither the site nor the nature of the mutation correlated with the severity of the clinical features. Severe truncations were found in different patients with either classical or early-onset forms of the disease.

Malfunction of nuclease ERCC1-XPF results in diverse clinical manifestations and causes Cockayne syndrome, xeroderma pigmentosum, and Fanconi anemia.

Cockayne syndrome (CS) is a genetic disorder characterized by developmental abnormalities and photodermatosis resulting from the lack of transcription-coupled nucleotide excision repair, which is responsible for the removal of photodamage from actively transcribed genes. To date, all identified causative mutations for CS have been in the two known CS-associated genes, ERCC8 (CSA) and ERCC6 (CSB). For the rare combined xeroderma pigmentosum (XP) and CS phenotype, all identified mutations are in three of the XP-associated genes, ERCC3 (XPB), ERCC2 (XPD), and ERCC5 (XPG). In a previous report, we identified several CS cases who did not have mutations in any of these genes. In this paper, we describe three CS individuals deficient in ERCC1 or ERCC4 (XPF). Remarkably, one of these individuals with XP complementation group F (XP-F) had clinical features of three different DNA-repair disorders--CS, XP, and Fanconi anemia (FA). Our results, together with those from Bogliolo et al., who describe XPF alterations resulting in FA alone, indicate a multifunctional role for XPF.

Xeroderma pigmentosum (complementation group D) mutation is present in patients affected by trichothiodystrophy with photosensitivity

SummaryWe studied the response to UV irradiation in cells from four patients, from three apparently unrelated families, affected by trichothiodystrophy (TTD). They showed all the symptoms of this rare autosomal recessive disorder (brittle hair with reduced sulfur content, mental and physical retardation, ichthyosis, peculiar face) together with photosensitivity. We found a decreased rate of duplicative DNA synthesis in stimulated lymphocytes, reduced survival in fibroblasts, and very low levels of unscheduled DNA synthesis (UDS) in Go lymphocytes and fibroblasts after UV irradiation. Complementation studies showed that normal values of UDS are restored in heterokaryons obtained by fusion of TTD cells with normal and xeroderma pigmentosum (XP)-complementation group A-cells. In contrast the defect is not complemented by fusion with XP-complementation group D-fibroblasts.

The role of CSA in the response to oxidative DNA damage in human cells

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5′S)-8,5′-cyclo 2′-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A

ABSTRACT Cells from complementation groups A through G of the heritable sun-sensitive disorder xeroderma pigmentosum (XP) show defects in nucleotide excision repair of damaged DNA. Proteins representing groups A, B, C, D, F, and G are subunits of the core recognition and incision machinery of repair. XP group E (XP-E) is the mildest form of the disorder, and cells generally show about 50% of the normal repair level. We investigated two protein factors previously implicated in the XP-E defect, UV-damaged DNA binding protein (UV-DDB) and replication protein A (RPA). Three newly identified XP-E cell lines (XP23PV, XP25PV, and a line formerly classified as an XP variant) were defective in UV-DDB binding activity but had levels of RPA in the normal range. The XP-E cell extracts did not display a significant nucleotide excision repair defect in vitro, with either UV-irradiated DNA or a uniquely placed cisplatin lesion used as a substrate. Purified UV-DDB protein did not stimulate repair of naked DNA by DDB− XP-E cell extracts, but microinjection of the protein into DDB−XP-E cells could partially correct the repair defect. RPA stimulated repair in normal, XP-E, or complemented extracts from other XP groups, and so the effect of RPA was not specific for XP-E cell extracts. These data strengthen the connection between XP-E and UV-DDB. Coupled with previous results, the findings suggest that UV-DDB has a role in the repair of DNA in chromatin.

Mutations in UVSSA cause UV-sensitive syndrome and impair RNA polymerase IIo processing in transcription-coupled nucleotide-excision repair

UV-sensitive syndrome (UVSS) is a genodermatosis characterized by cutaneous photosensitivity without skin carcinoma. Despite mild clinical features, cells from individuals with UVSS, like Cockayne syndrome cells, are very UV sensitive and are deficient in transcription-coupled nucleotide-excision repair (TC-NER), which removes DNA damage in actively transcribed genes. Three of the seven known UVSS cases carry mutations in the Cockayne syndrome genes ERCC8 or ERCC6 (also known as CSA and CSB, respectively). The remaining four individuals with UVSS, one of whom is described for the first time here, formed a separate UVSS-A complementation group; however, the responsible gene was unknown. Using exome sequencing, we determine that mutations in the UVSSA gene (formerly known as KIAA1530) cause UVSS-A. The UVSSA protein interacts with TC-NER machinery and stabilizes the ERCC6 complex; it also facilitates ubiquitination of RNA polymerase IIo stalled at DNA damage sites. Our findings provide mechanistic insights into the processing of stalled RNA polymerase and explain the different clinical features across these TC-NER–deficient disorders.