Evidence for the role of EPHX2 gene variants in anorexia nervosa.

Anorexia nervosa (AN) and related eating disorders are complex, multifactorial neuropsychiatric conditions with likely rare and common genetic and environmental determinants. To identify genetic variants associated with AN, we pursued a series of sequencing and genotyping studies focusing on the coding regions and upstream sequence of 152 candidate genes in a total of 1205 AN cases and 1948 controls. We identified individual variant associations in the Estrogen Receptor-ß (ESR2) gene, as well as a set of rare and common variants in the Epoxide Hydrolase 2 (EPHX2) gene, in an initial sequencing study of 261 early-onset severe AN cases and 73 controls (P=0.0004). The association of EPHX2 variants was further delineated in: (1) a pooling-based replication study involving an additional 500 AN patients and 500 controls (replication set P=0.00000016); (2) single-locus studies in a cohort of 386 previously genotyped broadly defined AN cases and 295 female population controls from the Bogalusa Heart Study (BHS) and a cohort of 58 individuals with self-reported eating disturbances and 851 controls (combined smallest single locus P<0.01). As EPHX2 is known to influence cholesterol metabolism, and AN is often associated with elevated cholesterol levels, we also investigated the association of EPHX2 variants and longitudinal body mass index (BMI) and cholesterol in BHS female and male subjects (N=229) and found evidence for a modifying effect of a subset of variants on the relationship between cholesterol and BMI (P<0.01). These findings suggest a novel association of gene variants within EPHX2 to susceptibility to AN and provide a foundation for future study of this important yet poorly understood condition.

Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells.

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA.

Xeroderma pigmentosum complementation group C cells remove pyrimidine dimers selectively from the transcribed strand of active genes.

We have measured the removal of UV-induced pyrimidine dimers from DNA fragments of the adenosine deaminase (ADA) and dihydrofolate reductase (DHFR) genes in primary normal human and xeroderma pigmentosum complementation group C (XP-C) cells. Using strand-specific probes, we show that in normal cells, preferential repair of the 5' part of the ADA gene is due to the rapid and efficient repair of the transcribed strand. Within 8 h after irradiation with UV at 10 J m-2, 70% of the pyrimidine dimers in this strand are removed. The nontranscribed strand is repaired at a much slower rate, with 30% dimers removed after 8 h. Repair of the transcribed strand in XP-C cells occurs at a rate indistinguishable from that in normal cells, but the nontranscribed strand is not repaired significantly in these cells. Similar results were obtained for the DHFR gene. In the 3' part of the ADA gene, however, both normal and XP-C cells perform fast and efficient repair of either strand, which is likely to be caused by the presence of transcription units on both strands. The factor defective in XP-C cells is apparently involved in the processing of DNA damage in inactive parts of the genome, including nontranscribed strands of active genes. These findings have important implications for the understanding of the mechanism of UV-induced excision repair and mutagenesis in mammalian cells.

The sensitivity of Cockayne's syndrome cells to DNA-damaging agents is not due to defective transcription-coupled repair of active genes.

Two of the hallmarks of Cockayne's syndrome (CS) are the hypersensitivity of cells to UV light and the lack of recovery of the ability to synthesize RNA following exposure of cells to UV light, in spite of the normal repair capacity at the overall genome level. The prolonged repressed RNA synthesis has been attributed to a defect in transcription-coupled repair, resulting in slow removal of DNA lesions from the transcribed strand of active genes. This model predicts that the sensitivity of CS cells to another DNA-damaging agent, i.e., the UV-mimetic agent N-acetoxy-2-acetylaminofluorene (NA-AAF), should also be associated with a lack of resumption of RNA synthesis and defective transcription-coupled repair of NA-AAF-induced DNA adducts. We tested this by measuring the rate of excision of DNA adducts in the adenosine deaminase gene of primary normal human fibroblasts and two CS (complementation group A and B) fibroblast strains. High-performance liquid chromatography analysis of DNA adducts revealed that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the main adduct induced by NA-AAF in both normal and CS cells. No differences were found between normal and CS cells with respect to induction of this lesion either at the level of the genome overall or at the gene level. Moreover, repair of dG-C8-AF in the active adenosine deaminase gene occurred at similar rates and without strand specificity in normal and CS cells, indicating that transcription-coupled repair does not contribute significantly to repair of dG-C8-AF in active genes. Yet CS cells are threefold more sensitive to NA-AAF than are normal cells and are unable to recover the ability to synthesize RNA. Our data rule out defective transcription-coupled repair as the cause of the increased sensitivity of CS cells to DNA-damaging agents and suggest that the cellular sensitivity and the prolonged repressed RNA synthesis are primarily due to a transcription defect. We hypothesize that upon treatment of cells with either UV or NA-AAF, the basal transcription factor TFIIH becomes involved in nucleotide excision repair and that the CS gene products are involved in the conversion of TFIIH back to the transcription function. In this view, the CS proteins act as repair-transcription uncoupling factors. If the uncoupling process is defective, RNA synthesis will stay repressed, causing cellular sensitivity. Since transcription is essential for transcription-coupled repair, the CS defect will affect those lesions whose repair is predominantly transcription coupled, i.e., UV-induced cyclobutane pyrimidine dimers.

DNA strand specificity for UV-induced mutations in mammalian cells.

The influence of DNA repair on the molecular nature of mutations induced by UV light (254 nm) was investigated in UV-induced hprt mutants from UV-sensitive Chinese hamster cells (V-H1) and the parental line (V79). The nature of point mutations in hprt exon sequences was determined for 19 hprt mutants of V79 and for 17 hprt mutants of V-H1 cells by sequence analysis of in vitro-amplified hprt cDNA. The mutation spectrum in V79 cells consisted of single- and tandem double-base pair changes, while in V-H1 cells three frameshift mutations were also detected. All base pair changes in V-H1 mutants were due to GC----AT transitions. In contrast, in V79 all possible classes of base pair changes except the GC----CG transversion were present. In this group, 70% of the mutations were transversions. Since all mutations except one did occur at dipyrimidine sites, the assumption was made that they were caused by UV-induced photoproducts at these sites. In V79 cells, 11 out of 17 base pair changes were caused by photoproducts in the nontranscribed strand of the hprt gene. However, in V-H1 cells, which are completely deficient in the removal of pyrimidine dimers from the hprt gene and which show a UV-induced mutation frequency enhanced seven times, 10 out of 11 base pair changes were caused by photoproducts in the transcribed strand of the hprt gene. We hypothesize that this extreme strand specificity in V-H1 cells is due to differences in fidelity of DNA replication of the leading and the lagging strand. Furthermore, we propose that in normal V79 cells two processes determine the strand specificity of UV-induced mutations in the hprt gene, namely preferential repair of the transcribed strand of the hprt gene and a higher fidelity of DNA replication of the nontranscribed strand compared with the transcribed strand.

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage.

In this study the role of nucleotide excision repair (NER) in protecting mouse embryonic stem (ES) cells against the genotoxic effects of UV-photolesions was analysed. Repair of cyclobutane pyrimidine dimers (CPD) in transcribed genes could not be detected whereas the removal of (6-4) photoproducts (6-4PP) was incomplete, already reaching its maximum (30%) 4 h after irradiation. Measurements of repair replication revealed a saturation of NER activity at UV doses >5 J/m2 while at a lower dose (2.5 J/m2) the repair kinetics were similar to those in murine embryonic fibroblasts (MEFs). Cytotoxic and mutagenic effects of photolesions were determined in ES cells differing in NER activity. ERCC1-deficient ES cells were hypermutable (10-fold) compared to wild-type cells, indicating that at physiologically relevant doses ES cells efficiently remove photolesions. The effect of the NER deficiency on cytoxicity was only 2-fold. Exposure to high UV doses (10 J/m2) resulted in a rapid and massive induction of apoptosis. Possibly, to avoid the accumulation of mutated cells, ES cells rely on the induction of a strong apoptotic response with a simultaneous shutting down of NER activity.

Molecular analysis of ethyl methanesulfonate-induced mutations at the hprt gene in the ethyl methanesulfonate-sensitive Chinese hamster cell line EM-C11 and its parental line CHO9.

The Chinese hamster cell line EM-C11 has been shown to be 5 times more sensitive than its parental line CHO9, but not hypermutable, after treatment with ethyl methanesulfonate. Ethyl methanesulfonate-induced mutational spectra were determined at the hprt locus to investigate whether the same adducts are responsible for mutation induction in both cell lines. The mutational spectra for EM-C11 and CHO9 show an important difference. GC-->AT transitions were found in both cell lines at similar frequencies; however, the spectrum of CHO9 contains a class of AT-->GC transitions, which seems to be replaced by a group of deletions in EM-C11. Since the ethyl methanesulfonate-induced mutation frequency for both lines is the same at equal exposure, it is hypothesized that the lesions leading to AT-->GC transitions in CHO9 are responsible for the deletions in EM-C11. This phenomenon might be explained if the responsible adduct(s) in CHO9 is bypassed resulting in replication errors, while blocking DNA synthesis in EM-C11 causing the observed increase in cell death. In surviving EM-C11 cells, DNA strand exchanges might have occurred at the position of stalled replication forks, leading to gross molecular changes. The adduct probably responsible for the AT-->GC transitions in CHO9 and the deletions in EM-C11 is 3-ethyladenine.

Mutagenic processing of ethylation damage in mammalian cells: the use of methoxyamine to study apurinic/apyrimidinic site-induced mutagenesis.

The aldehyde reagent methoxyamine is able to interact with apurinic/apyrimidinic sites formed in vivo within cells and displays both an anti-cytotoxic and an antimutagenic activity on N-ethyl-N'-nitro-N-nitrosoguanidine-induced DNA damage in Chinese hamster ovary cells. To clarify the underlying mechanism we have examined the mutational spectra induced by N-ethyl-N'-nitro-N-nitrosoguanidine alone and in the presence of methoxyamine in the hypoxanthine-guanine phosphoribosyltransferase gene of Chinese hamster ovary cells. In both cases all mutations were base pair substitutions, and their distribution among various classes did not differ significantly. Almost 60% were transitions, predominantly GC to AT, and the remaining 40% were transversions, mainly at AT base pairs. The analysis of the proportion of the different types of mutations showed that in the presence of methoxyamine, GC to AT transitions decreased by a factor of 1.8, and AT to CG transversions were reduced by a factor of 13. These data indicate that in mammalian cells the fixation of ethylation damage into mutations occurs by both (a) direct mutagenesis likely driven by O6-ethylguanine adducts and to a minor extent by O4-ethylthymine and (b) apurinic/apyrimidinic site-mediated mutagenesis. These apurinic/apyrimidinic sites are formed during the processing of ethylation at critical sites and are likely to involve O6-ethylguanine and O2-ethylthymine adducts.

Impairment of nucleotide excision repair by apoptosis in UV-irradiated mouse cells.

We investigated the relationship between nucleotide excision repair (NER) activity and apoptosis in UV-irradiated cells. Mouse erythroleukemia (MEL) and lymphoma (GRSL) cells exhibited enhanced sensitivity to the cytotoxic effects of UV radiation compared to hamster cell lines, although normal UV-induced hprt mutation frequencies were found. Determination of UV-induced repair replication revealed a limited capacity of MEL and GRSL cells to perform NER consistent with poor removal of cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts from transcriptionally active genes during the first 8 h after UV exposure. However, both cyclobutane pyrimidine dimers and pyrimidine 6-4 pyrimidone photoproducts appeared to be processed to almost normal level 24 h after UV treatment. In parallel, we observed that the UV-irradiated MEL and GRSL cells suffered from severe DNA fragmentation particularly 24 h after UV exposure. Taken together, these data indicate a reduced repair of UV-induced photolesions in apoptotic cells, already established at the early onset of apoptosis. To test whether inhibition of repair in cells was due to inactivation of NER or to apoptosis-induced chromatin degradation, we performed in vitro excision assays using extracts from UV-irradiated MEL cells. These experiments showed that the NER capacity during early apoptosis was intact, indicating that slow removal of UV-induced photolesions in apoptotic cells is due to substrate modification (presumably degradation of chromatin) rather than direct inhibition of factors involved in NER.

Molecular analysis of hprt gene mutations in skin fibroblasts of rats exposed in vivo to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

The granuloma pouch assay in the rat is a model system in which relative frequencies of genetic and (pre-) neoplastic changes induced in vivo by carcinogenic agents can be determined within the same target tissue. The target is granuloma pouch tissue and consists of a population of (transient) proliferating fibroblasts which can be cultured in vitro. hprt gene mutations were studied in granuloma pouch tissue of rats treated with single doses of direct acting alkylating agents N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an exposure-dependent increase in the hprt mutant frequency. Thirty-seven MNU (60 mg/kg)- and 43 ENU (100 mg/kg)-induced hprt mutant cell clones were analyzed at the molecular level. Twenty-two MNU-induced and 36 ENU-induced mutants carried a single base pair change in exon sequences of the hprt gene. The predominant base pair alterations induced by MNU were GC to AT transitions (18 of 22), which are probably caused by O6-methylguanine lesions. For most of the GC to AT transitions (16 of 18), the G was located in the nontranscribed strand, suggesting a strand bias in the repair of O6-methylguanine lesions. ENU-induced mutations occurred predominantly at AT base pairs (28 of 36), being mostly AT to TA and AT to CG transversions, and are probably caused by O2-ethylthymidine. Also here, DNA repair processes seem to act with different rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since all the 24 transversions observed at AT base pairs had the thymidine residue in the nontranscribed strand. GC to AT transitions were only present at a low frequency among ENU-induced mutations, suggesting that O6-ethylguanine lesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU- and ENU-induced hprt mutant clones were different from spontaneously occurring hprt mutant clones. These results indicate that MNU and ENU induce different mutational spectra in vivo and that DNA repair systems remove O6-methylguanine, O2, and/or O4-ethylthymidine much faster from the transcribed strand than the nontranscribed strand of the hprt gene in these rat fibroblasts.

Ultraviolet-induced cyclobutane pyrimidine dimers are selectively removed from transcriptionally active genes in the epidermis of the hairless mouse.

This study describes the induction and repair of UV-induced cyclobutane pyrimidine dimers (CPD) in transcriptionally active and inactive genes in the epidermis of the hairless mouse. Mice were exposed to a single dose of 2000 J/m2 ultraviolet B and kept in darkness for up to 24 h. The CPD frequency was measured in the transcriptionally active hypoxanthine-guanine phosphoribosyltransferase gene, the adenosine deaminase gene, the inactive c-mos protooncogene, and the haptoglobin gene using the CPD-specific enzyme T4 endonuclease V. Sixty % of the CPD was removed from the active genes during the first 4 h, after which no further repair took place up to 24 h. In contrast, the inactive genes did not show any removal of CPD. Assuming that the rate of repair in the c-mos and haptoglobin genes is representative for the repair rate in the genome overall, these results suggest only marginal repair of UV-induced CPD in the mouse epidermis in vivo. The selective repair of active genes in the epidermis of mice resembles that of rodent cells in culture and shows the biological relevance of repair studies performed with cultured rodent cells in vitro.

Marked differences in the role of O6-alkylguanine in hprt mutagenesis in T-lymphocytes of rats exposed in vivo to ethylmethanesulfonate, N-(2-hydroxyethyl)-N-nitrosourea, or N-ethyl-N-nitrosourea.

The role of DNA alkylation at the O6 position of guanine in the induction of gene mutations in vivo was studied in the hprt gene of rat T-lymphocytes from spleen exposed in vivo to the monofunctional ethylating agents ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU), or the hydroxyethylating agent N-(2-hydroxyethyl)-N-nitrosourea (HOENU). All chemicals showed an exposure-dependent increase in hprt mutant frequency. HOENU and ENU, however, were much more mutagenic than EMS when compared at equimolar levels. DNA sequence analysis was performed on PCR products of hprt cDNA from 40 EMS-, 35 HOENU-, and 46 ENU-induced 6-thioguanine-resistant T-lymphocyte clones. Thirty EMS-induced mutants contained a single base pair substitution with GC to AT transitions being the predominant type of mutation (26 of 30) which are probably caused by mispairing of O6-ethylguanine with T during DNA replication. No strand specificity of mutated G's among GC to AT transitions was observed. Twenty-three HOENU- and 42 ENU-induced mutants contained a single base pair substitution. In contrast to EMS, GC to AT transitions were found at a low frequency, 4 of 23 for HOENU and 5 of 42 for ENU, indicating that O6-hydroxyethylguanine and O6-ethylguanine are less important in HOENU- and ENU-induced mutagenesis in vivo, respectively. Also here no strand bias for mutated G's was observed, although the number of this type of mutation was limited. The most frequently induced base pair alterations by HOENU and ENU were transversions at AT base pairs, 16 of 23 and 28 of 42, respectively, with AT to TA being the predominant type of mutation. In both ENU and HOENU mutational spectra, an extreme strand bias for mutated T's toward the nontranscribed strand was found. The results suggest that DNA damage induced in rat T-lymphocytes in vivo by HOENU and ENU is processed in similar ways.

Elevated frequencies of benzo(a)pyrene-induced Hprt mutations in internal tissue of XPA-deficient mice.

Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight and have a high predisposition to developing cancer. At the cellular level, XP patients are defective in nucleotide excision repair (NER). Recently, mice have been generated via gene targeting that are deficient in the expression of the XPA gene [A. de Vries et al., Nature (Lond.), 377: 169-173, 1995]. We have assessed the consequences of defective NER for mutagenesis in normal and XPA mice exposed to benzo(a)pyrene and 2-acetylaminofluorene. To study mutagenesis, mature T lymphocytes were isolated from the spleen and stimulated to proliferate in vitro to select for mutants at the endogenous Hprt locus. Background mutant frequencies in normal and XPA mice were very similar and not influenced by age. Single doses of benzo(a)pyrene administered i.p. resulted in a dose-dependent increase of the Hprt mutant frequency in normal mice. In addition, after chronic exposure to benzo(a)pyrene, Hprt mutants were readily detectable in XPA mice at an early onset of treatment but only at a later stage in normal mice. In contrast, chronic treatment of either normal or XPA mice with 2-acetylaminofluorene did not increase Hprt mutant frequency above the background frequency. This absence of significant induction of Hprt mutants can be entirely attributed to the low frequency of 2-acetylaminofluorene-induced DNA adducts in lymphoid tissue. These results provide the first direct evidence in mammals that deficient NER leads to enhanced mutagenesis in endogenous genes in internal tissue after exposure to relevant environmental mutagens, such as benzo(a)pyrene.

The relationship between benzo[a]pyrene-induced mutagenesis and carcinogenesis in repair-deficient Cockayne syndrome group B mice.

Cockayne syndrome (CS) patients are deficient in the transcription coupled repair (TCR) subpathway of nucleotide excision repair (NER) but in contrast to xeroderma pigmentosum patients, who have a defect in the global genome repair subpathway of NER, CS patients do not have an elevated cancer incidence. To determine to what extent a TCR deficiency affects carcinogen-induced mutagenesis and carcinogenesis, CS group B correcting gene (CSB)-deficient mice were treated with the genotoxic carcinogen benzo(a)pyrene (B[a]P) at an oral dose of 13 mg/kg body weight, three times a week. At different time points, mutant frequencies at the inactive lacZ gene (in spleen, liver, and lung) as well as at the active hypoxanthine phosphoribosyltransferase (Hprt) gene (in spleen) were determined to compare mutagenesis at inactive versus active genes. B[a]P treatment gave rise to increased mutant frequencies at lacZ in all of the organs tested without a significant difference between CSB-/- and wild-type mice, whereas B[a]P-induced Hprt mutant frequencies in splenic T-lymphocytes were significantly more enhanced in CSB-/- mice than in control mice. The sequence data obtained from Hprt mutants indicate that B[a]P adducts at guanine residues were preferentially removed from the transcribed strand of the Hprt gene in control mice but not in CSB-/- mice. On oral treatment with B[a]P, the tumor incidence increased in both wild-type and CSB-deficient animals. However, no differences in tumor rate were observed between TCR-deficient CSB-/- mice and wild-type mice, which is in line with the normal cancer susceptibility of CS patients. The mutagenic response at lacZ, in contrast to Hprt, correlated well with the cancer incidence in CSB-/- mice after B[a]P treatment, which suggests that mutations in the bulk of the DNA (inactive genes) are a better predictive marker for carcinogen-induced tumorigenesis than mutations in genes that are actively transcribed. Thus, the global genome repair pathway of NER appears to play an important role in the prevention of cancer.

Strand-specific removal of cyclobutane pyrimidine dimers from the p53 gene in the epidermis of UVB-irradiated hairless mice.

Removal of UVB-induced cyclobutane pyrimidine dimers (CPD) from each of the two strands of the transcriptionally active p53 tumor suppressor gene and the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was determined in the epidermis of the hairless mouse using the CPD-specific enzyme T4 endonuclease V. Mice were exposed to a single dose of UVB (2 kJ/m2) and kept in darkness for up to 24 h. About 80% of the CPD were removed from the transcribed strand of the p53 and HPRT genes within 24 h. Most rapid removal was observed during the first 4 h. In contrast, very little removal of CPD from the nontranscribed strand of the p53 and the HPRT genes was observed in 24 h. The same low level of repair was observed in the inactive c-mos proto-oncogene. The efficient repair of the transcribed strand compared to the nontranscribed strand of transcriptionally active genes in the epidermis of the hairless mouse resembles the repair of CPD in cultured rodent cells. Moreover, the selective removal of CPD from the transcribed strand of the p53 gene correlates well with the known strand bias of u.v.-induced mutations at dipyrimidine sites in the p53 gene of u.v.-induced mouse skin tumors.

Age-dependent spontaneous mutagenesis in Xpc mice defective in nucleotide excision repair.

DNA damages caused by cellular metabolites and environmental agents induce mutations, that may predispose to cancer. Nucleotide excision repair (NER) is a major cellular defence mechanism acting on a variety of DNA lesions. Here, we show that spontaneous mutant frequencies at the Hprt gene increased 30-fold in T-lymphocytes of 1 year old Xpc-/- mice, possessing only functional transcription-coupled repair (TCR). Hprt mutant frequencies in Xpa-/- and Csb-/- mice that both have a defect in this NER subpathway, remained low during ageing. In contrast to current models, the elevated mutation rate in Xpc-/- mice does not lead to an increased tumour incidence or premature ageing. Oncogene (2000) 19, 5034 - 5037

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells.

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.

Analysis of the structure and spatial distribution of ultraviolet-induced DNA repair patches in human cells made in the presence of inhibitors of replicative synthesis.

The repair of ultraviolet-induced damage in the presence of hydroxyurea or hydroxyurea and arabinosylcytosine was investigated in confluent human fibroblasts at the level of DNA loops attached to the nuclear matrix. Estimation of single-strand break frequencies based on the release of DNA from the DNA-nuclear matrix complex after incubation with nuclease S1 revealed the occurrence of multiple incision events per DNA loop in the presence of inhibitors. When both inhibitors were employed, over 90% of the repair-labelled DNA was not ligated within 2 h post-incubation. In the absence of ligation of repair patches, we observed a preferential release of repair-labelled DNA from the nuclear matrix by nuclease S1 compared to prelabelled DNA, regardless of the period of post-UV incubation. The results suggest that repair events are clustered to some extent in a certain area of a DNA loop. However, the position of these clusters relative to the attachment sites of DNA loops at the nuclear matrix is random. The data are discussed in terms of denaturation of a putative repair complex in the presence of hydroxyurea resulting in an excess of incisions over repaired sites.

Spectrum of spontaneously occurring mutations in the hprt gene of V79 Chinese hamster cells.

A total of 76 independent spontaneous mutants in the hprt gene of V79 Chinese hamster cells have been analyzed. These mutants were obtained in two different laboratories, 17 and 59 mutants in sets 1 and 2, respectively, under different cell culture conditions. Mutation analysis was performed by amplification of hprt cDNA with the polymerase chain reaction and direct sequencing of the products. The data obtained showed similar spectra of spontaneous mutations in both sets of mutants, suggesting that culture does not play a major role in spontaneous mutagenesis. The majority of the mutations were base substitutions (greater than 60%), with twice as many transversions as transitions. Base changes were evenly distributed throughout the structural gene, including the splice junctions. All types of base substitutions appeared in comparable frequencies, except for A.T to T.A transversions, which were almost absent. The fraction of deletion mutations was low (13%). A striking feature of the observed mutation spectra is that one third of the spontaneous mutations analyzed involved aberrant splicing of the hprt primary transcript, with exon 4 being affected most frequently, indicating that splice mutations are a common mechanism of mutation in the hprt gene.