NORMAL GENOMIC

• Cleaver JE
• Cancer in xeroderma pigmentosum and related disorders of DNA repair.
• Nat Rev Cancer. 2005; 5: 564-73
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• Nucleotide-excision repair diseases exhibit cancer, complex developmental disorders and neurodegeneration. Cancer is the hallmark of xeroderma pigmentosum (XP), and neurodegeneration and developmental disorders are the hallmarks of Cockayne syndrome and trichothiodystrophy. A distinguishing feature is that the DNA-repair or DNA-replication deficiencies of XP involve most of the genome, whereas the defects in CS are confined to actively transcribed genes. Many of the proteins involved in repair are also components of dynamic multiprotein complexes, transcription factors, ubiquitylation cofactors and signal-transduction networks. Complex clinical phenotypes might therefore result from unanticipated effects on other genes and proteins.

• Magnaldo T, Sarasin A
• Xeroderma pigmentosum: from symptoms and genetics to gene-based skin therapy.
• Cells Tissues Organs. 2004; 177: 189-98
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• Xeroderma pigmentosum (XP) is a rare, recessively inherited genodermatosis prone to ultraviolet (UV)-induced skin neoplasms from keratinocyte origin, i.e. basal and squamous cell carcinoma. Cells from classic XP patients fail to properly eliminate UV-induced DNA lesions by the nucleotide excision repair (NER) mechanism. A variant form of XP, called XP-V suffers from faulty translesion synthesis. We review here recent data on XP gene products whose alterations affect NER and result in one of the 7 complementation groups of XP. Encouraging results of retrovirus-based genetic correction of XP keratinocytes are summarized and support realistic prospects of gene therapy for the XP-C complementation group.

• Koeppel F, Poindessous V, Lazar V, Raymond E, Sarasin A, Larsen AK
• Irofulven cytotoxicity depends on transcription-coupled nucleotide excision repair and is correlated with XPG expression in solid tumor cells.
• Clin Cancer Res. 2004; 10: 5604-13
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• BACKGROUND: Irofulven is a novel alkylating agent with promising clinical activity, particularly toward ovarian and hormone-refractory prostate cancers. To facilitate additional clinical development, we have aimed to identify biological markers associated with sensitivity to the compound. METHODS: Fibroblasts derived from patients with xeroderma pigmentosum or Cockayne's syndrome along with a panel of 20 human cancer cell lines (eight different tumor types) were examined to establish the importance of nucleotide excision repair proteins in the sensitivity to irofulven. RESULTS: Human cells deficient in nucleotide excision repair are up to 30-fold more sensitive to the cytotoxic effects of irofulven compared with repair-proficient controls, clearly indicating that nucleotide excision repair plays a crucial role in the sensitivity to the drug. Interestingly, our results show that irofulven-induced lesions are recognized by transcription-coupled repair but not by global genome repair. Another unique feature is the pronounced sensitivity of XPD and XPB helicase-deficient cells to the drug. Comparison of the IC50 values for irofulven, cisplatin, and ecteinascidin 743 with the expression levels of ERCC1, XPD, and XPG genes in different solid tumor cell lines shows no correlation between the expression levels of any of the three nucleotide excision repair proteins and the sensitivity to ecteinascidin 743. In contrast, expression of the XPG endonuclease was correlated with the cytotoxicity for irofulven and, to a lesser degree, for cisplatin. Importantly, XPG expression was also correlated with cellular nucleotide excision repair activity. CONCLUSIONS: Increasing evidence indicates that compromised nucleotide excision repair activity is frequent in several solid tumor types. The results presented here suggest that XPG expression in such tumors may be a useful marker to predict their sensitivity to irofulven.

• Zhou NY, Bates SE, Bouziane M, Stary A, Sarasin A, O'Connor TR
• Efficient repair of cyclobutane pyrimidine dimers at mutational hot spots is restored in complemented Xeroderma pigmentosum group C and trichothiodystrophy/xeroderma pigmentosum group D cells.
• J Mol Biol. 2003; 332: 337-51
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• Xeroderma pigmentosum (XP) and trichothiodystrophy (TTD) are rare heritable diseases. Patients suffering from XP and 50% of TTD afflicted individuals are photosensitive and have a high susceptibility to develop skin tumors. One solution to alleviating symptoms of these diseases is to express the deficient cDNAs in patient cells as a form of gene therapy. XPC and TTD/XPD cell lines were complemented using retroviral transfer. Expressed wild-type XPC or XPD cDNAs in these cells restored the survival to UVC radiation to wild-type levels in the respective complementation groups. Although complemented XP cell lines have been studied for years, data on cyclobutane pyrimidine dimer (CPD) repair in these cells at different levels are sparse. We demonstrate that CPD repair is faster in the complemented lines at the global, gene, strand specific, and nucleotide specific levels than in the original lines. In both XPC and TTD/XPD complemented lines, CPD repair on the non-transcribed strand is faster than that for the MRC5SV line. However, global repair in the complemented cell lines and MRC5SV is still slower than in normal human fibroblasts. Despite the slower global repair rate, in the complemented XPC and TTD/XPD cells, almost all of the CPDs at "hotspots" for mutation in the P53 tumor database are repaired as rapidly as in normal human fibroblasts. Such evaluation of repair at nucleotide resolution in complemented nucleotide excision repair deficient cells presents a crucial way to determine the efficient re-establishment of function needed for successful gene therapy, even when full repair capacity is not restored.

• Svetlova M et al.
• Clustered sites of DNA repair synthesis during early nucleotide excision repair in ultraviolet light-irradiated quiescent human fibroblasts.
• Exp Cell Res. 2002; 276: 284-95
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• The ubiquitous process of nucleotide excision repair includes an obligatory step of DNA repair synthesis (DRS) to fill the gapped heteroduplex following excision of a short (approximately 30-nucleotide) damaged single-strand fragment. Using 5-iododeoxyuridine to label repair patches during the first 10-60 min after UV irradiation of quiescent normal human fibroblasts we have visualized a limited number of discrete foci of DRS. These must reflect clusters of elementary DRS patches, since single patches would not be detected. The DRS foci are attenuated in normal cells treated with alpha-amanitin or in Cockayne syndrome (CS) cells, which are specifically deficient in the pathway of transcription-coupled repair (TCR). It is therefore likely that the clusters of DRS arise in chromatin domains within which RNA polymerase II transcription is compartmentalized. However, we also found significant suppression of DRS foci in xeroderma pigmentosum, complementation group C cells in which global genome repair (GGR) is defective, but TCR is normal. This suggests that the TCR is responsible for the DRS cluster formation in the absence of GGR. The residual foci detected in CS cells indicate that, even at early times following UV irradiation, GGR may open some chromatin domains for processive scanning and consequent DRS independent of transcription.

• Moriwaki S, Kraemer KH
• Xeroderma pigmentosum--bridging a gap between clinic and laboratory.
• Photodermatol Photoimmunol Photomed. 2001; 17: 47-54
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• Xeroderma pigmentosum (XP) is an autosomal recessive photosensitive disorder with an extremely high incidence of UV-related skin cancers associated with impaired ability to repair UV-induced DNA damage. There are seven nucleotide excision repair (NER) complementation groups (A through G) and an NER proficient form (XP variant). XPA, B, D and G patients may also develop XP neurological disease. The laboratory diagnosis of XP can be performed by autoradiography. Recently, the isolation and characterization of the genes responsible for XP have made it possible to use molecular biological techniques to diagnose XP patients, for carrier detection and for prenatal diagnosis, especially in Japanese XPA patients. These techniques include polymerase chain reaction (PCR) and plasmid host cell reactivation assays with cloned XP genes. DNA damage is not repaired by the NER system equally throughout the genome. There are two DNA repair pathways: 1) transcription-coupled repair, and 2) global genome repair. Many factors involved in these pathways are related to the pathogenesis of XP and a related photosensitive disease, Cockayne syndrome. Clinical management consists of early diagnosis followed by a rigorous program of sun protection including avoidance of unnecessary UV exposure, wearing UV blocking clothing, and use of sunblocks on the skin. Although there is no cure for XP, the efficacy of oral retinoids for the prevention of new skin cancers, local injection of interferon, and the external use of a prokaryotic DNA repair enzyme have been reported.

• Graham JM Jr et al.
• Cerebro-oculo-facio-skeletal syndrome with a nucleotide excision-repair defect and a mutated XPD gene, with prenatal diagnosis in a triplet pregnancy.
• Am J Hum Genet. 2001; 69: 291-300
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• Cerebro-oculo-facio-skeletal (COFS) syndrome is a recessively inherited rapidly progressive neurologic disorder leading to brain atrophy, with calcifications, cataracts, microcornea, optic atrophy, progressive joint contractures, and growth failure. Cockayne syndrome (CS) is a recessively inherited neurodegenerative disorder characterized by low to normal birth weight, growth failure, brain dysmyelination with calcium deposits, cutaneous photosensitivity, pigmentary retinopathy and/or cataracts, and sensorineural hearing loss. Cultured CS cells are hypersensitive to UV radiation, because of impaired nucleotide-excision repair (NER) of UV-induced damage in actively transcribed DNA, whereas global genome NER is unaffected. The abnormalities in CS are caused by mutated CSA or CSB genes. Another class of patients with CS symptoms have mutations in the XPB, XPD, or XPG genes, which result in UV hypersensitivity as well as defective global NER; such patients may concurrently have clinical features of another NER syndrome, xeroderma pigmentosum (XP). Clinically observed similarities between COFS syndrome and CS have been followed by discoveries of cases of COFS syndrome that are associated with mutations in the XPG and CSB genes. Here we report the first involvement of the XPD gene in a new case of UV-sensitive COFS syndrome, with heterozygous substitutions-a R616W null mutation (previously seen in patients in XP complementation group D) and a unique D681N mutation-demonstrating that a third gene can be involved in COFS syndrome. We propose that COFS syndrome be included within the already known spectrum of NER disorders: XP, CS, and trichothiodystrophy. We predict that future patients with COFS syndrome will be found to have mutations in the CSA or XPB genes, and we document successful use of DNA repair for prenatal diagnosis in triplet and singleton pregnancies at risk for COFS syndrome. This result strongly underlines the need for screening of patients with COFS syndrome, for either UV sensitivity or DNA-repair abnormalities.

• Tuteja N, Tuteja R
• Unraveling DNA repair in human: molecular mechanisms and consequences of repair defect.
• Crit Rev Biochem Mol Biol. 2001; 36: 261-90
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• Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders.

• Berneburg M, Lehmann AR
• Xeroderma pigmentosum and related disorders: defects in DNA repair and transcription.
• Adv Genet. 2001; 43: 71-102
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• The genetic disorders xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD) are all associated with defects in nucleotide excision repair (NER) of DNA damage. Their clinical features are very different, however, XP being a highly cancer-prone skin disorder, whereas CS and TTD are cancer-free multisystem disorders. All three are genetically complex, with at least eight complementation groups for XP (XP-A to -G and variant), five for CS (CS-A, CS-B, XP-B, XP-D, and XP-G), and three for TTD (XP-B, XP-D, and TTD-A). With the exception of the variant, the products of the XP genes are proteins involved in the different steps of NER, and comprise three damage-recognition proteins, two helicases, and two nucleases. The two helicases, XPB and XPD, are components of the basal transcription factor TFIIH, which has a dual role in NER and initiation of transcription. Different mutations in these genes can affect NER and transcription differentially, and this accounts for the different clinical phenotypes. Mutations resulting in defective repair without affecting transcription result in XP, whereas if transcription is also affected, TTD is the outcome. CS proteins are only involved in transcription-coupled repair, a subpathway of NER in which damage in the transcribed strands of active genes is rapidly and preferentially repaired. Current evidence suggests that they also have an important but not essential role in transcription. The variant form of XP is defective in a novel DNA polymerase, which is able to synthesise DNA past UV-damaged sites.

• Egly JM
• The 14th Datta Lecture. TFIIH: from transcription to clinic.
• FEBS Lett. 2001; 498: 124-8
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• 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.

• de Boer J, Hoeijmakers JH
• Nucleotide excision repair and human syndromes.
• Carcinogenesis. 2000; 21: 453-60
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• DNA damage is implicated in cancer and aging, and several DNA repair mechanisms exist that safeguard the genome from these deleterious consequences. Nucleotide excision repair (NER) removes a wide diversity of lesions, the main of which include UV-induced lesions, bulky chemical adducts and some forms of oxidative damage. The NER process involves the action of at least 30 proteins in a 'cut-and-paste'-like mechanism. The consequences of a defect in one of the NER proteins are apparent from three rare recessive syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and the photosensitive form of the brittle hair disorder trichothiodystrophy (TTD). Sun-sensitive skin is associated with skin cancer predisposition in the case of XP, but remarkably not in CS and TTD. Moreover, the spectrum of clinical symptoms differs considerably between the three syndromes. CS and TTD patients exhibit a spectrum of neurodevelopmental abnormalities and, in addition, TTD is associated with ichthyosis and brittle hair. These typical CS and TTD abnormalities are difficult to comprehend as a consequence of defective NER. This review briefly describes the biochemistry of the NER process, summarizes the clinical features of the NER disorders and speculates on the molecular basis underlying these pleitropic syndromes.

• Winkler GS et al.
• TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair.
• J Biol Chem. 2000; 275: 4258-66
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• 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.

• Rapin I, Lindenbaum Y, Dickson DW, Kraemer KH, Robbins JH
• Cockayne syndrome and xeroderma pigmentosum.
• Neurology. 2000; 55: 1442-9
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• OBJECTIVES: To review genetic variants of Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive disorders of DNA repair that affect the nervous system, and to illustrate them by the first case of xeroderma pigmentosum-Cockayne syndrome (XP-CS) complex to undergo neuropathologic examination. METHODS: Published reports of clinical, pathologic, and molecular studies of CS, XP neurologic disease, and the XP-CS complex were reviewed, and a ninth case of XP-CS is summarized. RESULTS: CS is a multisystem disorder that causes both profound growth failure of the soma and brain and progressive cachexia, retinal, cochlear, and neurologic degeneration, with a leukodystrophy and demyelinating neuropathy without an increase in cancer. XP presents as extreme photosensitivity of the skin and eyes with a 1000-fold increased frequency of cutaneous basal and squamous cell carcinomas and melanomas and a small increase in nervous system neoplasms. Some 20% of patients with XP incur progressive degeneration of previously normally developed neurons resulting in cortical, basal ganglia, cerebellar, and spinal atrophy, cochlear degeneration, and a mixed distal axonal neuropathy. Cultured cells from patients with CS or XP are hypersensitive to killing by ultraviolet (UV) radiation. Both CS and most XP cells have defective DNA nucleotide excision repair of actively transcribing genes; in addition, XP cells have defective repair of the global genome. There are two complementation groups in CS and seven in XP. Patients with the XP-CS complex fall into three XP complementation groups. Despite their XP genotype, six of nine individuals with the XP-CS complex, including the boy we followed up to his death at age 6, had the typical clinically and pathologically severe CS phenotype. Cultured skin and blood cells had extreme sensitivity to killing by UV radiation, DNA repair was severely deficient, post-UV unscheduled DNA synthesis was reduced to less than 5%, and post-UV plasmid mutation frequency was increased. CONCLUSIONS: The paradoxical lack of parallelism of phenotype to genotype is unexplained in these disorders. Perhaps diverse mutations responsible for UV sensitivity and deficient DNA repair may also produce profound failure of brain and somatic growth, progressive cachexia and premature aging, and tissue-selective neurologic deterioration by their roles in regulation of transcription and repair of endogenous oxidative DNA damage.

• Tsutakawa SE, Cooper PK
• Transcription-coupled repair of oxidative DNA damage in human cells: mechanisms and consequences.
• Cold Spring Harb Symp Quant Biol. 2000; 65: 201-15

• van Hoffen A, Kalle WH, de Jong-Versteeg A, Lehmann AR, van Zeeland AA, Mullenders LH
• Cells from XP-D and XP-D-CS patients exhibit equally inefficient repair of UV-induced damage in transcribed genes but different capacity to recover UV-inhibited transcription.
• Nucleic Acids Res. 1999; 27: 2898-904
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• Xeroderma pigmentosum (XP) is a rare hereditary human disorder clinically associated with severe sun sensitivity and predisposition to skin cancer. Some XP patients also show clinical characteristics of Cockayne syndrome (CS), a disorder associated with defective preferential repair of DNA lesions in transcriptionally active genes. Cells from the two XP-patients who belong to complementation group D and exhibit additional clinical symptoms of CS are strikingly more sensitive to the cytotoxic effects of UV-light than cells from classical XP-D patients. To explain the severe UV-sensitivity it was suggested that XP-D-CS cells have a defect in preferential repair of UV-induced 6-4 photoproducts (6-4PP) in active genes. We investigated the capacity of XP-D and XP-D-CS cells to repair UV-induced DNA lesions in the active adenosine deaminase gene (ADA) and in the inactive 754 gene by determining (i) the removal of specific lesions, i.e. cyclobutane pyrimidine dimers (CPD) and 6-4PP, or (ii) the formation of BrdUrd-labeled repair patches. No differences in repair capacity were observed between XP-D and XP-D-CS cells. In both cell types repair of CPD was completely absent whereas 6-4PP were inefficiently removed from the ADA gene and the 754 gene with similar kinetics. However, whereas XP-D cells were able to restore UV-inhibited RNA synthesis after a UV-dose of 2 J/m2, RNA synthesis in XP-D-CS cells remained repressed up to 24 h after irradiation. Our results are inconsistent with the hypothesis that differences in the capacity to perform preferential repair of UV-induced photolesions in active genes between XP-D and XP-D-CS cells are the cause of the extreme UV-sensitivity of XP-D-CS cells. Rather, the enhanced sensitivity of XP-D-CS cells may be associated with a defect in transcription regulation superimposed on the repair defect.

• de Boer J et al.
• Mouse model for the DNA repair/basal transcription disorder trichothiodystrophy reveals cancer predisposition.
• Cancer Res. 1999; 59: 3489-94
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• Patients with the nucleotide excision repair (NER) disorder xeroderma pigmentosum (XP) are highly predisposed to develop sunlight-induced skin cancer, in remarkable contrast to photosensitive NER-deficient trichothiodystrophy (TTD) patients carrying mutations in the same XPD gene. XPD encodes a helicase subunit of the dually functional DNA repair/basal transcription complex TFIIH. The pleiotropic disease phenotype is hypothesized to be, in part, derived from a repair defect causing UV sensitivity and, in part, from a subtle, viable basal transcription deficiency accounting for the cutaneous, developmental, and the typical brittle hair features of TTD. To understand the relationship between deficient NER and tumor susceptibility, we used a mouse model for TTD that mimics an XPD point mutation of a TTD patient in the mouse germline. Like the fibroblasts from the patient, mouse cells exhibit a partial NER defect, evident from the reduced UV-induced DNA repair synthesis (residual repair capacity approximately 25%), limited recovery of RNA synthesis after UV exposure, and a relatively mild hypersensitivity to cell killing by UV or 7,12-dimethylbenz[a]anthracene. In accordance with the cellular studies, TTD mice exhibit a modestly increased sensitivity to UV-induced inflammation and hyperplasia of the skin. In striking contrast to the human syndrome, TTD mice manifest a dear susceptibility to UV- and 7,12-dimethylbenz[a]anthracene-induced skin carcinogenesis, albeit not as pronounced as the totally NER-deficient XPA mice. These findings open up the possibility that TTD is associated with a so far unnoticed cancer predisposition and support the notion that a NER deficiency enhances cancer susceptibility. These findings have important implications for the etiology of the human disorder and for the impact of NER on carcinogenesis.

• Coin F, Bergmann E, Tremeau-Bravard A, Egly JM
• Mutations in XPB and XPD helicases found in xeroderma pigmentosum patients impair the transcription function of TFIIH.
• EMBO J. 1999; 18: 1357-66
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• As part of TFIIH, XPB and XPD helicases have been shown to play a role in nucleotide excision repair (NER). Mutations in these subunits are associated with three genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). The strong heterogeneous clinical features observed in these patients cannot be explained by defects in NER alone. We decided to look at the transcriptional activity of TFIIH from cell lines of XP individuals. We set up an immunopurification procedure to isolate purified TFIIH from patient cell extracts. We demonstrated that mutations in two XP-B/CS patients decrease the transcriptional activity of the corresponding TFIIH by preventing promoter opening. The defect of XPB in transcription can be circumvented by artificial opening of the promoter. Western blot analysis and enzymatic assays indicate that XPD mutations affect the stoichiometric composition of TFIIH due to a weakness in the interaction between XPD-CAK complex and the core TFIIH, resulting in a partial reduction of transcription activity. This work, in addition to clarifying the role of the various TFIIH subunits, supports the current hypothesis that XP-B/D patients are more likely to suffer from transcription repair syndromes rather than DNA repair disorders alone.

• Leadon SA
• Transcription-coupled repair of DNA damage: unanticipated players, unexpected complexities.
• Am J Hum Genet. 1999; 64: 1259-63

• Rapic Otrin V et al.
• Relationship of the xeroderma pigmentosum group E DNA repair defect to the chromatin and DNA binding proteins UV-DDB and replication protein A.
• Mol Cell Biol. 1998; 18: 3182-90
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• 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.

• Quilliet X et al.
• Retroviral-mediated correction of DNA repair defect in xeroderma pigmentosum cells is associated with recovery of catalase activity.
• Mutat Res. 1997; 385: 235-42
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• Xeroderma pigmentosum (XP) is a rare inherited disease associated with photosensitivity, a very high susceptibility to develop neoplasm on sun-exposed skin and neurological abnormalities for some patients. We previously reported that diploid cell lines established from XP skin biopsies present an abnormal low level of catalase activity, which is involved in the defense against oxygen free radicals. This biochemical dysfunction, probably involved in the skin cancer formation, has been difficult to be directly related to the nucleotide excision repair (NER) defect in XP. In this paper we report that the retroviral-mediated transduction of XP diploid cells by the XPC and XPD/ERCC2 cDNAs fully and stably corrects the NER defect in terms of survival and unscheduled DNA synthesis (UDS) after ultraviolet (UV) irradiation. The catalase activity in transduced cells was recovered up to normal levels only in cells transduced with repair genes correcting the repair defect. These results imply that: (i) the reduced catalase activity in XP, which might result from cellular depletion of its NADPH cofactor, is directly related to impaired DNA repair, and (ii) this depletion might be one of the multiple cellular consequences of XP inborn defect.

• Sarasin A, Stary A
• Human cancer and DNA repair-deficient diseases.
• Cancer Detect Prev. 1997; 21: 406-11
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• Cancer development requires the accumulation of numerous genetic changes which are usually believed to occur through the presence of unrepaired DNA lesions. Exogenous or endogenous DNA-damaging agents can lead to mutations in the absence of efficient error-free repair, via replication of DNA damage. Several DNA repair pathways are present in living cells and well conserved from bacteria to human cells. Apart from mismatch repair, photolyases, base excision, and postreplication repair, the nucleotide excision repair (NER), the most versatile of these DNA repair systems, recognizes and eliminates a wide variety of DNA lesions and particularly those induced by ultraviolet (UV) light. The phenotypic consequences of an NER defect in humans are apparent in rare but dramatic diseases characterized by hypersensitivity to UV and a striking clinical and genetic heterogeneity. The xeroderma pigmentosum syndrome (XP), the Cockayne's syndrome (CS), and the photosensitive form of trichothiodystrophy (TTD) are three of these clinically distinct human disorders inherited as an autosomal recessive trait. Persistence of unrepaired DNA damage produced by exposure to UV light is associated, in the XP syndrome, with an extremely high level of skin tumors in sun-exposed sites. But the direct link of defective DNA repair to cancer seems to be complex, since, in contrast to patients with XP, those with TTD or CS do not have an increased frequency of skin cancers. The understanding of the absence of skin tumors in TTD and CS patients may offer a way to better protect normal individuals from the most rapidly increasing cancer: skin cancer.

• Kazantsev A, Mu D, Nichols AF, Zhao X, Linn S, Sancar A
• Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.
• Proc Natl Acad Sci U S A. 1996; 93: 5014-8
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• Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.

• Boulikas T
• Xeroderma pigmentosum and molecular cloning of DNA repair genes.
• Anticancer Res. 1996; 16: 693-708
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• Human cells from patients suffering with xeroderma pigmentosum (XP) characterized by extreme sensitivity to UV light and a high incidence of skin tumors fall into seven complementation groups, XPA to XPG, and are lacking a functional helicase, endonuclease, or lesion-recognizing protein involved in the initial steps during nucleotide excision repair (NER); a number of proteins involved in DNA repair are termed XPA to XPG depending on which one is defective in a particular complementation group of XP and include: (i) proteins involved in the recognition of (6-4) photoproducts (XPE) and of a broad range of lesions such as pyrimidine dimers (XPA); (ii) proteins that are DNA helicases and integral parts of the general transcription factor TFIIH functioning in both transcription and repair (XPB, XPD); (iii) endonucleases that perform the two incisions, the XPG incising six nucleotides (nt) to the 3' side from a photodimer and the ERCC1-XPF protein complex incising 22 nt to the 5' side of the lesion; and (iv) single-strand DNA-binding proteins (XPC). The ERCC6 helicase is largely responsible for coupling transcription to repair whereas XPC seems to be responsible for the repair of the inactive parts of the genome as well as for the repair of the nontranscribed strand in active genes. p53 recognizes insertion/deletion mismatches as well as free ends of DNA produced by ionizing radiation to arrest the cell cycle. Most of the human DNA repair proteins have their counterparts in both budding and fission yeasts and some of them also in E. coli evoking an evolutionary conservation of DNA repair pathways. Accumulation of mutations within repair genes in single cells followed by their escape from the immune surveillance and in clonal expansion may greatly contribute to the appearance and development of human cancers.

• Jaspers NG
• Multiple involvement of nucleotide excision repair enzymes: clinical manifestations of molecular intricacies.
• Cytokines Mol Ther. 1996; 2: 115-9
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• Nucleotide excision repair (NER) is a process required to remove DNA damage inflicted upon our skin by the short-wave bands of natural sunlight. Defective NER may result in a high risk of UV-induced skin tumors, since it occurs in patients with the inherited disorder xeroderma pigmentosum (XP). However, Cockayne's syndrome (CS) and PIBIDS (a photosensitive form of trichothiodystrophy) are also disorders with defective NER, but show no evidence of an elevated risk of cancer. In addition, many of CS and PIBIDS symptoms are difficult to explain on the basis of an NER defect only. Recent new insights into the molecular mechanisms of NER have shown additional involvements of many NER enzymes in other cellular processes. These multiple functions are likely to be the basis of the complex symptomatology of XP, CS and PIBIDS. Specific gene-targeted mouse models will probably help to solve these intricacies.

• Vermeulen W et al.
• Clinical heterogeneity within xeroderma pigmentosum associated with mutations in the DNA repair and transcription gene ERCC3.
• Am J Hum Genet. 1994; 54: 191-200
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• The human DNA excision repair gene ERCC3 specifically corrects the nucleotide excision repair (NER) defect of xeroderma pigmentosum (XP) complementation group B. In addition to its function in NER, the ERCC3 DNA helicase was recently identified as one of the components of the human BTF2/TFIIH transcription factor complex, which is required for initiation of transcription of class II genes. To date, a single patient (XP11BE) has been assigned to this XP group B (XP-B), with ther remarkable conjunction of two autosomal recessive DNA repair deficiency disorders: XP and Cockayne syndrome (CS). The intriguing involvement of the ERCC3 protein in the vital process of transcription may provide an explanation for the rarity, severity, and wide spectrum of clinical features in this complementation group. Here we report the identification of two new XP-B patients: XPCS1BA and XPCS2BA (siblings), by microneedle injection of the cloned ERCC3 repair gene as well as by cell hybridization. Molecular analysis of the ERCC3 gene in both patients revealed a single base substitution causing a missense mutation in a region that is completely conserved in yeast, Drosophila, mouse, and human ERCC3. As in patient XP11BE, the expression of only one allele (paternal) is detected. The mutation causes a virtually complete inactivation of the NER function of the protein. Despite this severe NER defect, both patients display a late onset of neurologic impairment, mild cutaneous symptoms, and a striking absence of skin tumors even at an age of > 40 years. Analysis of the frequency of hprt- mutant T-lymphocytes in blood samples suggests a relatively low in vivo mutation frequency in these patients. Factors in addition to NER deficiency may be required for the development of cutaneous tumors.

• Keeney S et al.
• Correction of the DNA repair defect in xeroderma pigmentosum group E by injection of a DNA damage-binding protein.
• Proc Natl Acad Sci U S A. 1994; 91: 4053-6
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• Cells from a subset of patients with the DNA-repair-defective disease xeroderma pigmentosum complementation group E (XP-E) are known to lack a DNA damage-binding (DDB) activity. Purified human DDB protein was injected into XP-E cells to test whether the DNA-repair defect in these cells is caused by a defect in DDB activity. Injected DDB protein stimulated DNA repair to normal levels in those strains that lack the DDB activity but did not stimulate repair in cells from other xeroderma pigmentosum groups or in XP-E cells that contain the activity. These results provide direct evidence that defective DDB activity causes the repair defect in a subset of XP-E patients, which in turn establishes a role for this activity in nucleotide-excision repair in vivo.

• Stefanini M et al.
• A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy.
• Am J Hum Genet. 1993; 53: 817-21
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• The sun-sensitive, cancer-prone genetic disorder xeroderma pigmentosum (XP) is associated in most cases with a defect in the ability to carry out excision repair of UV damage. Seven genetically distinct complementation groups (i.e., A-G) have been identified. A large proportion of patients with the unrelated disorder trichothiodystrophy (TTD), which is characterized by hair-shaft abnormalities, as well as by physical and mental retardation, are also deficient in excision repair of UV damage. In most of these cases the repair deficiency is in the same complementation group as is XP group D. We report here on cells from a patient, TTD1BR, in which the repair defect complements all known XP groups (including XP-D). Furthermore, microinjection of various cloned human repair genes fails to correct the repair defect in this cell strain. The defect in TTD1BR cells is therefore in a new gene involved in excision repair in human cells. The finding of a second DNA repair gene that is associated with the clinical features of TTD argues strongly for an involvement of repair proteins in hair-shaft development.

• Wood RD et al.
• Nucleotide excision repair of DNA by mammalian cell extracts and purified proteins.
• Cold Spring Harb Symp Quant Biol. 1993; 58: 625-32

• Satoh MS, Jones CJ, Wood RD, Lindahl T
• DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions.
• Proc Natl Acad Sci U S A. 1993; 90: 6335-9
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• Plasmid DNA was gamma-irradiated or treated with H2O2 in the presence of Cu2+ to generate oxygen free radical-induced lesions. Open circular DNA molecules were removed by ethidium bromide/CsCl density gradient centrifugation. The closed circular DNA fraction was treated with the Escherichia coli reagent enzymes endonuclease III (Nth protein) and Fpg protein. This treatment converted DNA molecules containing the major base lesions pyrimidine hydrates and 8-hydroxyguanine to a nicked form. Remaining closed circular DNA containing other oxygen radical-induced base lesions was used as a substrate for nucleotide excision-repair in a cell-free system. Extracts from normal human cells, but not extracts from xeroderma pigmentosum cells, catalyzed repair synthesis in this DNA. The repair defect in the latter extracts could be specifically corrected by in vitro complementation. The data suggest that accumulation of endogenous oxidative damage in cellular DNA from xeroderma pigmentosum patients contributes to the increased frequency of internal cancers and the neural degeneration occurring in serious cases of the syndrome.

• O'Donovan A, Wood RD
• Identical defects in DNA repair in xeroderma pigmentosum group G and rodent ERCC group 5.
• Nature. 1993; 363: 185-8
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• Humans with the complementation group G form of the inherited syndrome xeroderma pigmentosum (XP) are hypersensitive to solar ultraviolet light because of a defect in nucleotide-excision repair of DNA. Some individuals are also affected with Cockayne's syndrome, and have neurological abnormalities. Here we report that the DNA repair deficiency of XP-G cell extracts can be corrected by addition of protein fractions from normal cells. Repair proficiency can also be restored by mixing XP-G cell extracts with extracts from different repair-defective cell lines, with one exception. Extracts from cells representing group 5 of a set of ultraviolet-sensitive rodent mutants fail to complement XP-G extracts. XP-G and group 5 correcting activities co-elute after approximately 1,000-fold purification from HeLa cells. An antibody directed against a recombinant fragment of the XP-G complementing protein (XPGC) inhibits excision repair by normal cell extracts, and activity can be restored with an XP-G/group 5 complementing fraction. These data strongly suggest that the XPGC and group 5 correcting (ERCC5) proteins are identical.