Defects in the DNA repair and transcription gene ERCC2(XPD) in trichothiodystrophy (original) (raw)
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Carcinogenesis, 1993
Trichothiodystrophy (TTD) is a rare genetic disease associated in-50% of patients with DNA repair deficiency analogous to that found in xeroderma pigmentosum group D (XP-D) patients. Although XP-D patients exhibit a very high level of skin cancer on sun-exposed parts, TTD is not associated with cancer. We analysed UV-induced mutations in TTD cells and compared them to data in XP-D in order to determine if the molecular mechanisms of mutagenesis can explain the discrepancies between these two syndromes. We first immortalized a fibroblast TTD line with an ori~-SV40 plasmid. To investigate the kinds of mutations induced in TTD cells, we used an UV-irradiated (at 254 nm) shuttle vector carrying the supF tRNA gene as a target. We compared our data with those published by others with the same pZ189 vector in normal and XP-D fibroblast lines (Bredberg et al.,
Cancer research, 1995
To understand the heterogeneity in genetic predisposition to skin cancer in different nucleotide excision repair-deficient human syndromes, we studied repair of cyclobutane pyrimidine dimers (CPDs) and of pyrimidine(6-4)pyrimidone (6-4PP) photoproducts in cells from trichothiodystrophy (TTD) patients. TTD is not associated with increased incidence of skin cancer, although 50% of the patients are photosensitive and carry a defect in the nucleotide excision repair pathway, similar to Xeroderma pigmentosum patients. However, in striking contrast to TTD, Xeroderma pigmentosum is highly prone to cancer. To address this apparent paradox, two types of studies were conducted: (a) reactivation of UV-irradiated plasmids harboring actively transcribed reporter genes, with or without photolyase treatment before transfection of SV40-transformed fibroblasts; and (b) the kinetics of removal of UV-induced CPDs and 6-4PPs in genomic DNA by immunoblot analysis using lesion-specific mAbs in SV40-trans...
Journal of Molecular Biology, 1995
To study the relationships between mutagenesis and carcinogenesis, we 1 Laboratory of Molecular compared the mutations and their frequency induced by ultraviolet Genetics, UPR 42 CNRS irradiation at 254 nm (UV-C) in XP-D (GM-08207B/XP6BE), TTD/XP-D IFC1, Institut de Recherches sur le Cancer, B.P. 8-94801 (TTD1VI-LAS-KMT11) and wild-type (MRC-5V1) human cells. XP-D and TTD/XP-D cells, mutated in the same XP-D/ERCC2 gene, are deficient in Villejuif, France nucleotide excision repair. Whereas XP-D patients develop early skin tumors, 2 Unit of Cancer Epidemiology TTD patients do not exhibit abnormal levels of cancers. U351 INSERM, Institut After verification of UV hypersensitivity and DNA repair defect of the Gustave Roussy, 39 rue immortalized cell lines XP-D and TTD compared with a wild-type cell line, Camille Desmoulins, 94805 UV-induced mutagenesis was studied with a new shuttle vector pR2, Villejuif cedex, France carrying the target lacZ' gene. The UV-mutation frequencies in XP-D and TTD cells were similar and significantly increased compared with normal cells. Sequence analysis of 312 independent mutant plasmids revealed that more rearrangements were induced in TTD cells (16%) than in XP-D (5%) and normal cells (1%), while XP-D cells exhibited a twofold higher rate of tandem mutations compared with TTD and normal cells. In the three cell lines, a predominance of G:C to A:T transitions was found, especially in XP-D cells (87%) and most mutations were targeted on dipyrimidine sites, chiefly on the cytosine at 5'-TC-3' sites. The types of UV-induced point mutations in TTD cells were, however, more similar to those in normal cells than those found in XP-D cells. XP-D mutations were preferentially located in 5'-TCPur-3' sites, while mutations in normal and TTD cells were mostly at 5'-TCC-3' sites. Analysis of mutation spectra revealed differences in the location of the mutational hotspots between the three lines. Although the mutation frequency of the UV-irradiated pR2 vector is much higher in TTD and XP-D cells than in normal cells, the mutation spectrum is closer between TTD and normal cells as compared with XP-D cells. These dissimilarities could contribute to an explanation of some of the differences between the two syndromes.
The American Journal of Human Genetics, 1998
Xeroderma pigmentosum (XP) complementation group D is a heterogeneous group, containing patients with XP alone, rare cases with both XP and Cockayne syndrome, and patients with trichothiodystrophy (TTD). TTD is a rare autosomal recessive multisystem disorder associated, in many patients, with a defect in nucleotideexcision repair; but in contrast to XP patients, TTD patients are not cancer prone. In most of the repairdeficient TTD patients, the defect has been assigned to the XPD gene. The XPD gene product is a subunit of transcription factor TFIIH, which is involved in both DNA repair and transcription. We have determined the mutations and the pattern of inheritance of the XPD alleles in the 11 cases identified in Italy so far, in which the hair abnormalities diagnostic for TTD are associated with different disease severity but similar cellular photosensitivity. We have identified eight causative mutations, of which four have not been described before, either in TTD or XP cases, supporting the hypothesis that the mutations responsible for TTD are different from those found in other pathological phenotypes. Arg112his was the most common alteration in the Italian patients, of whom five were homozygotes and two were heterozygotes, for this mutation. The presence of a specifically mutated XPD allele, irrespective of its homozygous, hemizygous, or heterozygous condition, was always associated with the same degree of cellular UV hypersensitivity. Surprisingly, however, the severity of the clinical symptoms did not correlate with the magnitude of the DNA-repair defect. The most severe clinical features were found in patients who appear to be functionally hemizygous for the mutated allele.
Cancer Research, 2008
Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFIIH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 6-4PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some TTD/XPD mutations affect the recruitment of TFIIH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFIIH complexes carrying an NH 2-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFIIH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFIIH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.
Differential Repair of the Two Major UV-Induced Photolesions in Trichothiodystrophy Fibroblasts
Cancer Research, 2004
Defects in nucleotide excision repair have been shown to be associated with the photosensitive form of the disorder trichothiodystrophy (TTD). Most repair-deficient TTD patients are mutated in the XPD gene, a subunit of the transcription factor TFIIH. Knowledge of the kinetics and efficiency of repair of the two major UV-induced photolesions in TTD is critical to understand the role of unrepaired lesions in the process of carcinogenesis and explain the absence of enhanced skin cancer incidence in TTD patients contrarily to the xeroderma pigmentosum D patients. In this study, we used different approaches to quantify repair of UV-induced cyclobutane pyrimidine dimers (CPD) and pyrimidine (6–4) pyrimidone photoproducts (6–4PP) at the gene and the genome overall level. In cells of two TTD patients, repair of CPD and 6–4PP was reduced compared with normal human cells, but the reduction was more severe in confluent cells than in exponentially growing cells. Moreover, the impairment of rep...
The American Journal of Human Genetics, 1995
Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are quite distinct genetic disorders that are associated with defects in excision repair of UV-induced DNA damage. A few patients have been described previously with the clinical features of both disorders. In this paper we describe an individual in this category who has unusual cellular responses to UV light. We show that his cultured fibroblasts and lymphocytes are extremely sensitive to irradiation with UV-C, despite a level of nucleotide excision repair that is 30% -40% that of normal cells. The deficiency is assigned to the XP-D complementation group, and we have identified two causative mutations in the XPD gene: a gly-).arg change at amino acid 675 in the allele inherited from the patient's mother and a -1 frameshift at amino acid 669 in the allele inherited from his father. These mutations are in the C-terminal 20% of the 760-amino-acid XPD protein, in a region where we have recently identified several mutations in patients with trichothiodystrophy.