Defects In Nucleotide Excision Repair Research Articles

Nucleotide excision repair assay in Drosophila melanogaster using established cell lines.

Nucleotide excision repair (NER) is one of the most important systems in eukaryotes for overcoming DNA damage caused by environmental agents, such as ultraviolet (UV) radiation and chemical mutagens. NER in eukaryotes has been studied by genetic analyses of mainly human, rodent, and yeast repairdeficient mutants (,). In particular, cultured cells derived from patients with xeroderma pigmentosum (XP), a human autosomal-recessive disease causing defects in NER, as well as from rodent excision repair crosscomplementing (ERCC) mutants have contributed to the elucidation of the molecular mechanisms of excision repair. Complementation analyses by cell fusion between cells from XP patients have defined seven complementation groups (A-G) and one variant (XP-V). With the exception of XPV and possibly XPE (see Chapters 7 and 9), these genes have been cloned and their counterparts from rodent and yeast identified (,). Elucidation of the molecular functions and interactions of these gene products have been advanced by the use of effective and powerful cell-free reconstitution systems. Wood et al. have developed a cell-free system utilizing UV-irradiated plasmid DNA and human cell extracts in which both UV-dependent NER and the differences between wild-type and XP cells are reproduced (; see Chapter 29). Functional complementation between cell extracts in this in vitro system has proven effective in the isolation and identification of new factors involved in NER ().

Molecular mechanism of nucleotide excision repair.

From its very beginning, life has faced the fundamental problem that the form in which genetic information is stored is not chemically inert. DNA integrity is challenged by the damaging effect of numerous chemical and physical agents, compromizing its function. To protect this Achilles heel, an intricate network of DNA repair systems has evolved early in evolution. One of these is nucleotide excision repair (NER), a highly versatile and sophisticated DNA damage removal pathway that counteracts the deleterious effects of a multitude of DNA lesions, including major types of damage induced by environmental sources. The most relevant lesions subject to NER are cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4PPs), two major kinds of injury produced by the shortwave UV component of sunlight. In addition, numerous bulky chemical adducts are eliminated by this process. Within the divergent spectrum of NER lesions, significant distortion of the DNA helix appears to be a common denominator. Defects in NER underlie the extreme photosensitivity and predisposition to skin cancer observed with the prototype repair syndrome xeroderma pigmentosum (XP). Seven XP complementation groups have been identified, representing distinct repair genes XPA–G (discussed in detail below). In the last decade, all key NER factors have been cloned and the core of the ‘cut-and-paste’ reaction has been reconstituted in vitro from purified components. Recently, XPC (complexed to hHR23B) has been identified as a DNA-damage sensor and repair-recruitment factor. The general transcription factor complex TFIIH, containing the XPB and XPD helicases, mediates strand separation at the site of the lesion. XPA verifies the damage in an open DNA conformation and is crucial in the assembly of the remainder of the repair machinery. Replication protein A (RPA) stabilizes the opened DNA complex and is involved in positioning the XPG and ERCC1–XPF endonucleases responsible for the DNA incisions around the lesion. After removal of the damagecontaining oligonucleotide, typically 24–32 nucleotides in length, general replication factors fill in the remaining gap and close it. Two modes of NER can be distinguished: repair of lesions over the entire genome, referred to as global genome NER (GG–NER), and repair of transcription-blocking lesions present in transcribed DNA strands, hence called transcription-coupled NER (TC–NER). Most XP groups harbor defects in a common component of both NER subpathways. GG–NER is dependent on the activity of all factors mentioned above, including the GG– NER-specific complex XPC–hHR23B. The rate of repair for GG–NER strongly depends on the type of lesion. For instance, 6-4PPs are removed much faster from the genome than CPDs, probably because of differences in affinity of the damage sensor XPC–hHR23B. In addition, the location (accessibility) of a lesion influences the removal rate in vivo. In TC–NER, damage is detected by the elongating RNA polymerase II complex when it encounters a lesion. Interestingly, a distinct disorder, Cockayne syndrome (CS), is associated with a specific defect in transcription-coupled repair. The identification of two complementation groups (CS-A and CS-B) shows that at least two gene products are specifically needed for fast and efficient repair of transcribed strands. Phenotypically, CS is a very pleiotropic condition characterized by photosensitivity as well as severe neurological, developmental, and premature aging features. Most of these symptoms are not seen even with totally NERdeficient XP patients. The additional symptoms of CS suggest that transcription-coupled repair and/or the CS proteins have functions beyond NER. Also, non-NERspecific lesions (such as oxidative damage) that stall transcription elongation appear to be removed in a transcription-coupled fashion, linking a blocked polymerase to multiple repair pathways. Intriguingly, some XP-B, XP-D, and XP-G patients display CS features combined with XP manifestations. Yet other XP-B and XP-D individuals suffer from the CS-like brittle-hair syndrome trichothiodystrophy (TTD). This clinical conundrum points to additional roles of these NER factors as well. A recent mouse model for TTD has linked mutations in the XPD subunit of the dual functional TFIIH complex with deficiencies in basal transcription underlying at least some of the TTD manifestations. Thus, NER defects are associated with a surprisingly wide clinical heterogeneity due to additional functions of the NER factors involved. 1Corresponding author. E-MAIL Hoeijmakers@gen.fgg.eur.nl; FAX 31 10 408 9468.

Analysis of Mutations in the XPD Gene in Italian Patients with Trichothiodystrophy: Site of Mutation Correlates with Repair Deficiency, but Gene Dosage Appears to Determine Clinical Severity

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 nucleotide-excision repair; but in contrast to XP patients, TTD patients are not cancer prone. In most of the repair-deficient 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.

Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH.

In most cases, xeroderma pigmentosum group D (XP-D) and trichothiodystrophy (TTD) patients carry mutations in the carboxy-terminal domain of the evolutionarily conserved helicase XPD, which is one of the subunits of the transcription/repair factor TFIIH (refs 1,2). In this study, we demonstrate that XPD interacts specifically with p44, another subunit of TFIIH, and that this interaction results in the stimulation of 5'-->3' helicase activity. Mutations in the XPD C-terminal domain, as found in most patients, prevent the interaction with p44, thus explaining the decrease in XPD helicase activity and the nucleotide excision repair (NER) defect.

Aberrant p21WAF1-dependent growth arrest as the possible mechanism of abnormal resistance to ultraviolet light cytotoxicity in Li-Fraumeni syndrome fibroblast strains heterozygous for TP53 mutations.

The purpose of this study is to better understand the roles of the p53 tumor suppressor protein and the product of the p53-regulated gene p21WAF1 in the response of diploid human dermal fibroblast cultures to 254 nm ultraviolet (UV) light. We report that Li-Fraumeni syndrome (LFS) fibroblast strains heterozygous for TP53 mutation at either codon 245 or 234 exhibit markedly reduced or no expression of p21WAF1 following UV irradiation, respectively. These strains also exhibit defective nucleotide excision repair and pronounced inhibition of RNA synthesis following UV exposure, both of which are molecular hallmarks of cells derived from patients with the UV-sensitive syndrome xeroderma pigmentosum. In sharp contrast to xeroderma pigmentosum cells, however, the repair-deficient LFS cells show abnormal resistance, rather than hypersensitivity, to the killing effect of UV light. We further demonstrate that exposure of normal human fibroblasts to biologically relevant fluences (< or = 15 J/m2) of UV does not induce apoptotic cell death, indicating that UV resistant phenotype displayed by LFS strains is not associated with deregulated apoptosis. In normal fibroblasts, such treatment results in a moderate ( threefold) up-regulation of p53 protein, induction of the p21WAF1 gene, and a senescence-like growth arrest. On the other hand, exposure to > or = 20 J/m2 UV results in a striking up-regulation of p53, inhibition of p21WAF1 expression, and activation of an apoptotic pathway. We conclude that: (i) p21WAF1-mediated senescence is the principal mode of cell death induced by < or = 15 J/m2 UV light in normal human fibroblasts; (ii) there is a threshold effect for p53-dependent apoptosis and that, in normal human cells, this threshold level is induced upon expsoure to 20 J/m2 UV; (iii) the p53 signaling pathway is malfunctional in the TP53 heterozygous LFS strains examined; and (iv) the enhanced resistance to UV-induced cell killing displayed by these LFS strains is a consequence of diminished growth arrest, which is presumably mediated by p21WAF1 and not abnormalities in an apoptotic pathway.

Saccharomyces cerevisiae exonuclease-1 plays a role in UV resistance that is distinct from nucleotide excision repair.

Two closely related genes, EXO1 and DIN 7, in the budding yeast Saccharomyces cerevisiae have been found to be sequence homologs of the exo1 gene from the fission yeast Schizosaccharomyces pombe . The proteins encoded by these genes belong to the Rad2/XPG and Rad27/FEN-1 families, which are structure-specific nucleases functioning in DNA repair. An XPG nuclease deficiency in humans is one cause of xeroderma pigmentosum and those afflicted display a hypersensitivity to UV light. Deletion of the RAD2 gene in S. cerevisiae also causes UV hypersensitivity, due to a defect in nucleotide excision repair (NER), but residual UV resistance remains. In this report, we describe evidence for the residual repair of UV damage to DNA that is dependent upon Exo1 nuclease. Expression of the EXO1 gene is UV inducible. Genetic analysis indicates that the EXO1 gene is involved in a NER-independent pathway for UV repair, as exo1 rad2 double mutants are more sensitive to UV than either the rad2 or exo1 single mutants. Since the roles of EXO1 in mismatch repair and recombination have been established, double mutants were constructed to examine the possible relationship between the role of EXO1 in UV resistance and its roles in other pathways for repair of UV damaged DNA. The exo1 msh2 , exo1 rad51 , rad2 rad51 and rad2 msh2 double mutants were all more sensitive to UV than their respective pairs of single mutants. This suggests that the observed UV sensitivity of the exo1 deletion mutant is unlikely to be due to its functional deficiencies in MMR, recombination or NER. Further, it suggests that the EXO1 , RAD51 and MSH2 genes control independent mechanisms for the maintenance of UV resistance.

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

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.

A Mouse Model for the Basal Transcription/DNA Repair Syndrome Trichothiodystrophy

The sun-sensitive form of the severe neurodevelopmental, brittle hair disorder trichothiodystrophy (TTD) is caused by point mutations in the essential XPB and XPD helicase subunits of the dual functional DNA repair/basal transcription factor TFIIH. The phenotype is hypothesized to be in part derived from a nucleotide excision repair defect and in part from a subtle basal transcription deficiency accounting for the nonrepair TTD features. Using a novel gene-targeting strategy, we have mimicked the causative XPD point mutation of a TTD patient in the mouse. TTD mice reflect to a remarkable extent the human disorder, including brittle hair, developmental abnormalities, reduced life span, UV sensitivity, and skin abnormalities. The cutaneous symptoms are associated with reduced transcription of a skin-specific gene, strongly supporting the concept of TTD as a human disease due to inborn defects in basal transcription and DNA repair.

The transcription-repair coupling factor CSA is required for efficient repair only during the elongation stages of RNA polymerase II transcription

The known nucleotide excision repair (NER) defects of xeroderma pigmentosum (XP) and Cockayne syndrome (CS) cells can be exploited to analyze mechanisms of repair of UV-induced cyclobutane pyrimidine dimers (CPDs) at nucleotide (nt.) resolution. The two gene products of the CS complementation groups (CSA and CSB) have been implicated in the preferential repair of the transcribed strand of human genes. We had previously described very efficient repair of CPDs at sequences near the transcription initiation site of the human JUN gene in normal fibroblasts. Here, we have analyzed repair in a CSA fibroblast strain. CSA cells exhibited rapid repair near the transcription initiation site (positions −45 to +15) but were deficient in repair of sequences on the transcribed strand beginning around nt. +20. There was also no strand-selective repair of sequences further downstream of the start site (+260 to +450). The results suggest that the transcription-repair coupling factor (TRCF) CSA is required for efficient repair only during the elongation stages of RNA polymerase II transcription. We also discuss possible mechanisms of differential repair observed near the transcription initiation site in XP and CS cells and conclude that these in vivo repair data support some recent models obtained from nucleotide excision repair experiments in vitro.

DNA Structural Elements Required for ERCC1-XPF Endonuclease Activity

The heterodimeric complex ERCC1-XPF is a structure-specific endonuclease responsible for the 5' incision during mammalian nucleotide excision repair (NER). Additionally, ERCC1-XPF is thought to function in the repair of interstrand DNA cross-links and, by analogy to the homologous Rad1-Rad10 complex in Saccharomyces cerevisiae, in recombination between direct repeated DNA sequences. To gain insight into the role of ERCC1-XPF in such recombinational processes and in the NER reaction, we studied in detail the DNA structural elements required for ERCC1-XPF endonucleolytic activity. Recombinant ERCC1-XPF, purified from insect cells, was found to cleave stem-loop substrates at the DNA junction in the absence of other proteins like replication protein A, showing that the structure-specific endonuclease activity is intrinsic to the complex. Cleavage depended on the presence of divalent cations and was optimal in low Mn2+ concentrations (0.2 mM). A minimum of 4-8 unpaired nucleotides was required for incisions by ERCC1-XPF. Splayed arm and flap substrates were also cut by ERCC1-XPF, resulting in the removal of 3' protruding single-stranded arms. All incisions occurred in one strand of duplex DNA at the 5' side of a junction with single-stranded DNA. The exact cleavage position varied from 2 to 8 nucleotides away from the junction. One single-stranded arm, protruding either in the 3' or 5' direction, was necessary and sufficient for correct positioning of incisions by ERCC1-XPF. Our data specify the engagement of ERCC1-XPF in NER and allow a more direct search for its specific role in recombination.

Retrovirus-mediated DNA repair gene transfer into xeroderma pigmentosum cells: perspectives for a gene therapy.

The rare hereditary disease xeroderma pigmentosum (XP) is clinically characterized by extreme sun sensitivity and an increased predisposition for developing skin cancer. Cultured cells from XP patients exhibit hypersensitivity to ultraviolet (UV) radiation due to the defect in nucleotide excision repair (NER), and other cellular abnormalities. Seven genes identified in the classical XP forms, XPA to XPG, are involved in the NER pathway. In view of developing a strategy of gene therapy for XP, we devised recombinant retrovirus-carrying DNA repair genes for transfer and stable expression of these genes in cells from XP patients. Results showed that these retroviruses are efficient tools for transducing XP fibroblasts and correcting repair-defective cellular phenotypes by recovering normal UV survival, unscheduled DNA synthesis, and RNA synthesis after UV irradiation, and also other cellular abnormalities resulting from NER defects. These results imply that the first step of cellular gene therapy might be accomplished successfully.

Retroviral-mediated correction of DNA repair defect in xeroderma pigmentosum cells is associated with recovery of catalase activity

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.

Recruitment of the putative transcription-repair coupling factor CSB/ERCC6 to RNA polymerase II elongation complexes.

Cockayne's syndrome (CS) is a disease characterized by developmental and growth defects, sunlight sensitivity, and a defect in transcription-coupled nucleotide excision repair. The two principle proteins involved in CS, CSA and CSB/ERCC6, have been hypothesized to bind RNA polymerase II (Pol II) and link transcription to DNA repair. We have tested CSA and CSB in assays designed to determine their role in transcription-coupled repair. Using a unique oligo(dC)-tailed DNA template, we provide biochemical evidence that CSB/ERCC6 interacts with Pol II molecules engaged in ternary complexes containing DNA and nascent RNA. CSB is a DNA-activated ATPase, and hydrolysis of the ATP beta-gamma phosphoanhydride bond is required for the formation of a stable Pol II-CSB-DNA-RNA complex. Unlike CSB, CSA does not directly bind Pol II.

Identification and characterization of XPC-binding domain of hHR23B.

hHR23B was originally isolated as a component of a protein complex that specifically complements nucleotide excision repair (NER) defects of xeroderma pigmentosum group C cell extracts in vitro and was identified as one of two human homologs of the Saccharomyces cerevisiae NER gene product Rad23. Recombinant hHR23B has previously been shown to significantly stimulate the NER activity of recombinant human XPC protein (rhXPC). In this study we identify and functionally characterize the XPC-binding domain of hHR23B protein. We prepared various internal as well as terminal deletion products of hHR23B protein in a His-tagged form and examined their binding with rhXPC by using nickel-chelating Sepharose. We demonstrate that a domain covering 56 amino acids of hHR23B is required for binding to rhXPC as well as for stimulation of in vitro NER reactions. Interestingly, a small polypeptide corresponding to the XPC-binding domain is sufficient to exert stimulation of XPC NER activity. Comparison with known crystal structures and analysis with secondary structure programs provided strong indications that the binding domain has a predominantly amphipathic alpha-helical character, consistent with evidence that the affinity with XPC is based on hydrophobic interactions. Our work shows that binding to XPC alone is required and sufficient for the role of hHR23B in in vitro NER but does not rule out the possibility that the protein has additional functions in vivo.

Mechanism of open complex and dual incision formation by human nucleotide excision repair factors.

During nucleotide excision repair in human cells, a damaged DNA strand is cleaved by two endonucleases, XPG on the 3' side of the lesion and ERCC1-XPF on the 5' side. These structure-specific enzymes act at junctions between duplex and single-stranded DNA. ATP-dependent formation of an open DNA structure of approximately 25 nt around the adduct precedes this dual incision. We investigated the mechanism of open complex formation and find that mutations in XPB or XPD, the DNA helicase subunits of the transcription and repair factor TFIIH, can completely prevent opening and dual incision in cell-free extracts. A deficiency in XPC protein also prevents opening. The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation. In contrast, XPF or ERCC1-defective extracts open normally and generate a 3' incision, but fail to form the 5' incision. This same repair defect was observed in extracts from human xeroderma pigmentosum cells with an alteration in the C-terminal domain of XPB, suggesting that XPB has an additional role in facilitating 5' incision by ERCC1-XPF nuclease. These data support a mechanism in which TFIIH-associated helicase activity and XPC protein catalyze initial formation of the key open intermediate, with full extension to the cleavage sites promoted by the other core nucleotide excision repair factors. Opening is followed by dual incision, with the 3' cleavage made first.

Constancy of the Relative Biological Effectiveness of 42 MeV (p→Be + ) Neutrons among Cell Lines with Different DNA Repair Proficiencies

An important approach to understanding the role of the various DNA repair pathways in the cellular response to DNA-damaging agents is through the study of repair-deficient mutant cell lines. In the present study we used this strategy to assess the relative importance of four of these pathways for the repair of DNA damage induced by low-linear energy transfer (LET) gamma rays and intermediate-LET 42 MeV (p-->Be+) fast neutrons. The panel of hamster cell mutants that we characterized for their relative sensitivity to fast neutrons and gamma rays includes cell lines with defects in the nucleotide excision repair pathway; these can be further subdivided into mutants which are defective in nucleotide excision repair alone [UV5 (ERCC2-), UV24 (ERCC3-), UV135 (ERCC5-) and UV61 (ERCC6-)] compared to those which have an associated defect in the distinct but overlapping pathway for the repair of DNA crosslinks [UV20 (ERCC1-) and UV41 (ERCC4-)]. We also examined mutants with defects in the base excision repair pathway [EM9 (XRCC1-)] and the DNA-dependent protein kinase (DNA-PK)-mediated DNA double-strand break (DSB) repair pathway [xrs5 (XRCC5-)]. None of the mutants defective in nucleotide excision repair was differentially sensitized to fast neutrons or gamma rays; in fact, the slight radiosensitivity of these mutants under aerated conditions may be secondary to their defect in nucleotide excision repair. In contrast, deficiency in the base excision repair pathway resulted in a significant primary sensitization to both types of radiation (1.95-fold to gamma rays and 1.79-fold to neutrons). Deficiency in the DSB repair pathway mediated by DNA-PK resulted in a marked, but again similar, primary sensitization to gamma rays (4.2-fold) and neutrons (5.1-fold). Thus none of the repair pathways examined here exhibited a preferential role for the repair of damage induced by low-LET compared to intermediate-LET radiations; this resulted in an essentially constant relative biological effectiveness (RBE) of approximately 2 among the cell lines studied, independent of their DNA repair proficiency. However, consideration of these data along with data published previously for high-LET alpha particles suggests that, whereas the DNA-PK pathway is important for the repair of DSBs induced by low- and intermediate-LET radiations, it becomes less important as the LET increases beyond 100 keV/microm; thus this pathway may not be involved in repairing the more complex lesions induced by densely ionizing high-LET particles.

Incomplete complementation of the DNA repair defect in cockayne syndrome cells by the denV gene from bacteriophage T4 suggests a deficiency in base excision repair.

Endonuclease V (denV) from bacteriophage T4 has been examined for its ability to complement the repair defect in Cockayne syndrome (CS) cells of complementation groups A and B. CS is an autosomal recessive disorder characterized by hypersensitivity to UV light and a defect in the preferential repair of UV-induced lesions in transcriptionally active DNA by the nucleotide excision repair (NER) pathway. The denV gene was introduced into non-transformed normal and CS fibroblasts transiently via a recombinant adenovirus (Ad) vector and into SV40-transformed normal and CS cells via a retroviral vector. Expression of denV in CS-A cells resulted in partial correction of the UV-sensitive phenotype in assays of gene-specific repair and cell viability, while correction of CS-B cells by expression of denV in the same assays was minimal or non-existent. In contrast, denV expression led to enhanced host cell reactivation (HCR) of viral DNA synthesis in both CS complementation groups to near normal levels. DenV is a glycosylase which is specific for cyclobutane-pyrimidine dimers (CPDs) but does not recognize other UV-induced lesions. Previous work has indicated that CS cells can efficiently repair all non-CPD UV-induced transcription blocking lesions (S.F. Barrett et al.. Mutation Res. 255 (1991) 281-291 [1]) and that denV incised lesions are believed to be processed via the base excision repair (BER) pathway. The inability of denV to complement the NER defect in CS cells to normal levels implies an impaired ability to process denV incised lesions by the BER pathway, and suggests a role for the CS genes, particularly the CS-B gene, in BER.

O XV B.2 - O XV B.2 Nucleotide excision repair-defects and mutations in the XPA-deficient mice

O XV B.2 - O XV B.2 Nucleotide excision repair-defects and mutations in the XPA-deficient mice

B lymphocytes of xeroderma pigmentosum or Cockayne syndrome patients with inherited defects in nucleotide excision repair are fully capable of somatic hypermutation of immunoglobulin genes.

Recent experiments have strongly suggested that the process of somatic mutation is linked to transcription initiation. It was postulated that a mutator factor loads onto the RNA polymerase and, during elongation, causes transcriptional arrest that activates DNA repair, thus occasionally causing errors in the DNA sequence. We report the analysis of the role of one of the known DNA repair systems, nucleotide excision repair (NER), in somatic mutation. Epstein-Barrvirus-transformed B cells from patients with defects in NER (XP-B, XP-D, XP-V, and CS-A) were studied. Their heavy and light chain genes show a high frequency of point mutations in the variable (V), but not in the constant (C) regions. This suggests that these B cells can undergo somatic hypermutation despite significant defects in NER. Thus, it is doubtful that NER is an essential part of the mechanism of somatic hypermutation of Ig genes. As an aside, NER seems also not involved in Ig gene switch recombination.

Genes For Tfb2, Tfb3, and Tfb4 Subunits of Yeast Transcription/Repair Factor IIH: HOMOLOGY TO HUMAN CYCLIN-DEPENDENT KINASE ACTIVATING KINASE AND IIH SUBUNITS

Genes for the Tfb2, Tfb3, and Tfb4 subunits of yeast RNA polymerase transcription factor IIH (TFIIH) are described. All three genes are essential for cell viability, and antibodies against Tfb3 specifically inhibit transcription in vitro. A C-terminal deletion of Tfb2 caused a defect in nucleotide excision repair, as shown by UV sensitivity of the mutant strain and loss of nucleotide excision repair activity in cell extracts (restored by the addition of purified TFIIH). An interaction between Tfb3 and the Kin28 subunit of TFIIH was detected by the two-hybrid approach, consistent with a role for Tfb3 in linking kinase and core domains of the factor. The deduced amino acid sequence of Tfb2 is similar to that of the 52-kDa subunit of human TFIIH, while Tfb3 is identified as a RING finger protein homologous to the 36-kDa subunit of murine CAK (cyclin-dependent kinase activating kinase) and to the 32-kDa subunit of human TFIIH. Tfb4 is homologous to p34 of human TFIIH and is identified as the weakly associated 37-kDa subunit of the yeast factor. These and other findings reveal a one-to-one correspondence and high degree of sequence similarity between the entire set of yeast and human TFIIH polypeptides.