Restricted ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum cells.

A shuttle vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in cultured skin cells from a patient with the skin-cancer-prone, DNA repair-deficient disease xeroderma pigmentosum and in repair-proficient cells. After replication in the human cells, progeny plasmids were purified. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of Escherichia coli carrying a suppressible amber mutation in the beta-galactosidase gene. Plasmid survival in the xeroderma pigmentosum cells was less than that of pZ189 harvested from repair-proficient human cells. The point-mutation frequency in the 150-base-pair tRNA marker gene increased up to 100-fold with ultraviolet dose. Sequence analysis of 150 mutant plasmids revealed that mutations were infrequent at potential thymine-thymine dimer sites. Ninety-three percent of the mutant plasmids from the xeroderma pigmentosum cells showed G X C----A X T transitions, compared to 73% in the normal cells (P less than 0.002). There were significantly fewer transversions (P less than 0.002) (especially G X C----T X A) and multiple base substitutions (P less than 0.00001) than when pZ189 was passaged in repair-proficient cells. The subset of mutational changes that are common to ultraviolet-treated plasmids propagated in both repair-proficient and xeroderma pigmentosum skin cells may be associated with the development of ultraviolet-induced skin cancer in humans.     

Ultraviolet-induced mutations in Cockayne syndrome cells are primarily caused by cyclobutane dimer photoproducts while repair of other photoproducts is normal.

We compared the contribution to mutagenesis in Cockayne syndrome (CS) cells of the major class of UV photoproducts, the cyclobutane pyrimidine dimer, to that of other DNA photoproducts by using the mutagenesis shuttle vector pZ189. Lymphoblastoid cell lines from the DNA repair-deficient disorders CS and xeroderma pigmentosum (XP) and a normal line were transfected with UV-treated pZ189. Cyclobutane dimers were selectively removed before transfection by photoreactivation (PR), leaving nondimer photoproducts intact. After UV exposure and replication in CS and XP cells, plasmid survival was abnormally reduced and mutation frequency was abnormally elevated. After PR, plasmid survival increased and mutation frequency in CS cells decreased to normal levels but remained abnormal in XP cells. Sequence analysis of > 200 mutant plasmids showed that with CS cells a major mutational hot spot was caused by unrepaired cyclobutane dimers. These data indicate that with both CS and XP cyclobutane dimers are major photoproducts generating reduced plasmid survival and increased mutation frequency. However, unlike XP, CS cells are proficient in repair of nondimer photoproducts. Since XP but not CS patients have a high frequency of UV-induced skin cancers, our data suggest that prevention of UV-induce skin cancers is associated with proficient repair of nondimer photoproducts.     

Frequency of intrachromosomal homologous recombination induced by UV radiation in normally repairing and excision repair-deficient human cells.

To investigate the role of DNA damage and nucleotide excision repair in intrachromosomal homologous recombination, a plasmid containing duplicated copies of the gene coding for hygromycin resistance was introduced into the genome of a repair-proficient human cell line, KMST-6, and two repair-deficient lines, XP2OS(SV) from xeroderma pigmentosum complementation group A and XP2YO(SV) from complementation group F. Neither hygromycin-resistance gene codes for a functional enzyme because each contains an insertion/deletion mutation at a unique site, but recombination between the two defective genes can yield hygromycin-resistant cells. The rates of spontaneous recombination in normal and xeroderma pigmentosum cell strains containing the recombination substrate were found to be similar. The frequency of UV-induced recombination was determined for three of these cell strains. At low doses, the group A cell strain and the group F cell strain showed a significant increase in frequency of recombinants. The repair-proficient cell strain required 10- to 20-fold higher doses of UV to exhibit comparable increases in frequency of recombinants. These results suggest that unexcised DNA damage, rather than the excision repair process per se, stimulates such recombination.     

Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level.

The spectrum of spontaneous mutation of an endogenous mammalian cell gene has been determined at the DNA sequence level. Thirty independent spontaneous APRT- mutations were cloned and subsequently completely sequenced. Twenty-seven contained single base substitutions. Of these, 22 were G.C to A.T transitions, suggesting a major role for the deamination of cytosine in spontaneous mutagenesis of Chinese hamster ovary cells. The remaining mutants included a tandem double substitution, a -1 frameshift, and a 17-base-pair deletion flanked by a 2-base-pair direct repeat. Many of the independently recovered mutants were clustered at sites of multiple occurrence (hot spots). One site accounted for greater than 25% of all independently recovered events. Mutations were generally located within the coding sequence, although two mutations occurred within the consensus sequence for a 3' splice site.     

Ultraviolet light induction of diphtheria toxin-resistant mutants of normal and xeroderma pigmentosum human fibroblasts.

The UV induction of diphtheria toxin-resistant (DTr) mutants in normal and xeroderma pigmentosum human fibroblasts has been quantitatively characterized. A concentration of diphtheria toxin at which DTr cells are cross-resistant to Pseudomonas aeruginosa exotoxin A was determined and used in the selection of resistant mutants. Recovery of mutants was not influenced by the presence of wild-type cell densities of 1-8 x 10(5) per 9-cm plate, indicating no metabolic cooperation exists, in contrast to what is seen in the selection of some other variant phenotypes. Expression periods for UV-induced mutations differed with the severity of mutagen treatment and cell strain used. A relatively long (10-15 days after UV treatment) expression period was required for the maximum recovery of DTr mutants. Maximum recovery was followed by a decrease in mutation frequency on subsequent days evaluated. An apparent linear dose response within the dose range used was observed for UV-induced mutations in both normal and xeroderma pigmentosum fibroblasts. Our results indicate that xeroderma pigmentosum fibroblasts have higher UV-induced mutation frequencies per unit UV dose but similar frequencies per unit survival compared to normal cells within the range of UV doses tested.     

Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.

We have studied the molecular mechanisms of spontaneous mutations in mouse cells carrying a selectable bacterial gene. The mouse cells carry the Escherichia coli xanthine (guanine) phosphoribosyltransferase (gpt) gene in a retroviral shuttle vector integrated into chromosomal DNA in a proviral form. Cells with spontaneous mutations in the gpt gene were selected as resistant to 6-thioguanine and then were fused with COS cells for recovery of the mutant genes. Out of a total of 77 independent 6-thioguanine-resistant cell lines isolated in this study, vector sequences could be rescued from 43 of the mutant lines, and the base sequences were determined for the gpt genes in all 43 of these lines. There was a variety of mutational events among the mutant gpt genes sequenced. The most frequent mutational event was a deletion (in 29 of the 43 mutant genes), and the next most frequent event was a base substitution mutation (in 11 of the 43 mutant genes). Among the deletion mutants, the great majority represent deletions of less than 10 base pairs. In fact, 19 of the 29 deletion mutants had deletions of 3 base pairs, and among the mutants with 3-base-pair deletions, there was a very strong deletion hot spot appearing in 16 independent mutants. All 19 of the 3-base-pair deletions resulted in the "in frame" loss of an aspartic acid codon. Among the base substitution mutations, transitions and transversions occurred with approximately equal frequency. Our results raise the possibility that small deletions represent the predominant mechanisms by which spontaneous mutations occur in mammalian cells.     

Mutagenesis by third-strand-directed psoralen adducts in repair-deficient human cells: high frequency and altered spectrum in a xeroderma pigmentosum variant.

Psoralen-conjugated triple-helix-forming oligonucleotides have been used to generate site-specific mutations within mammalian cells. To investigate factors influencing the efficiency of oligonucleotide-mediated gene targeting, the processing of third-strand-directed psoralen adducts was compared in normal and repair-deficient human cells. An unusually high mutation frequency and an altered mutation pattern were seen in xeroderma pigmentosum variant (XPV) cells compared with normal, xeroderma pigmentosum group A (XPA), and Fanconi anemia cells. In XPV, targeted mutations were produced in the supF reporter gene carried in a simian virus 40 vector at a frequency of 30%, 3-fold above that in normal or Fanconi anemia cells and 6-fold above that in XPA. The mutations generated by targeted psoralen crosslinks and monoadducts in the XPV cells formed a pattern distinct from that in the other three cell lines, with mutations occurring not just at the damaged site but also at adjacent base pairs. Hence, the XPV cells may have an abnormality in trans-lesion bypass synthesis during repair and/or replication, implicating a DNA polymerase or an accessory factor as a basis of the defect in XPV. These results may help to elucidate the repair deficiency in XPV, and they raise the possibility that genetic manipulation via triplex-targeted mutagenesis may be enhanced by modulation of the XPV-associated activity in normal cells.     

Hepatitis B virus suppresses expression of human beta-interferon.

To determine whether hepatitis B virus (HBV) regulates the expression of the human beta-interferon gene, a series of recombinant bovine papilloma virus plasmids containing the human interferon gene and various fragments of the HBV genome were constructed and used to transform C127 cells, a murine fibroblast line. Analysis of the DNA from transformed C127 cells indicated that the interferon gene was intact and that the plasmids replicated as stable multicopy elements. The 1828-base-pair BamHI HBV DNA fragment containing the core antigen gene, but not the 2755-base-pair Bgl II HBV DNA fragment encoding both the surface antigen and the X antigen, suppressed the production of human beta-interferon. No effect by any of the recombinant plasmids on the synthesis of murine interferon was detected. The suppression of human beta-interferon by HBV occurs via a trans-acting factor. A frameshift mutation within the HBV core gene alleviates the inhibitory activity; thus we infer that the core protein is this factor or is crucially associated with this activity.     

A cell-free assay measuring repair DNA synthesis in human fibroblasts.

Osmotic disruption of confluent cultured human fibroblasts that have been irradiated or exposed to chemical carcinogens allows the specific measurement of repair DNA synthesis using dTTP as a precursor. Fibroblasts similarly prepared from various xeroderma pigmentosum cell lines show the deficiencies of UV-induced DNA synthesis predicted from in vivo studies, while giving normal responses to methyl methanesulfonate. A pyrimidine-dimer-specific enzyme, T4 endonuclease V, stimulated the rate of UV-induced repair synthesis with normal and xeroderma pigmentosum cell lines. This system should prove useful for identifying agents that induce DNA repair, and cells that respond abnormally to such induction. It should also be applicable to an in vitro complementation assay with repair-defective cells and proteins obtained from repair-proficient cells. Finally, by using actively growing fibroblasts and thymidine in the system, DNA replication can be measured and studied in vitro.     

Correction of xeroderma pigmentosum complementation group D mutant cell phenotypes by chromosome and gene transfer: involvement of the human ERCC2 DNA repair gene.

Cultured cells from individuals afflicted with the genetically heterogeneous autosomal recessive disorder xeroderma pigmentosum (XP) exhibit sensitivity to UV radiation and defective nucleotide excision repair. Complementation of these mutant phenotypes after the introduction of single human chromosomes from repair-proficient cells into XP cells has provided a means of mapping the genes involved in this disease. We now report the phenotypic correction of XP cells from genetic complementation group D (XP-D) by a single human chromosome designated Tneo. Detailed molecular characterization of Tneo revealed a rearranged structure involving human chromosomes 16 and 19, including the excision repair cross-complementing 2 (ERCC2) gene from the previously described human DNA repair gene cluster at 19q13.2-q13.3. Direct transfer of a cosmid bearing the ERCC2 gene conferred UV resistance to XP-D cells.     

Xeroderma pigmentosum neurological abnormalities correlate with colony-forming ability after ultraviolet radiation.

Xeroderma pigmentosum is an autosomal recessive disease in which DNA repair processes are defective. All xeroderma pigmentosum patients develop premature aging of sun-exposed skin, and some develop neurological abnormalities due to premature death of nerve cells. Sensitivity to ultraviolet radiation of 24 xeroderma pigmentosum fibroblast strains was studied in vitro by measuring each strain's ability to divide and form colonies after irradiation. The most sensitive strains were derived from patients who had an early onset of neurological abnormalities; less sensitive strains were from patients with a later onset; and the most resistant strains were from patients without neurological abnormalities. The UV sensitivities of strains from each member of a sibling pair with xeroderma pigmentosum were identical, indicating that UV sensitivity of xeroderma pigmentosum strains is determined by the patient's inherited DNA repair defect. The results suggest that effective DNA repair is required to maintain the functional integrity of the human nervous system by preventing premature death of neurons.     

Kinds of mutations formed when a shuttle vector containing adducts of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene replicates in human cells.

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPDE) replicates in human cells. A human embryonic kidney cell line, 293, was used as the eukaryotic host. The target gene for mutation analysis, supF, codes for a tyrosine suppressor tRNA and is strategically located between the origin of replication of the plasmid in Escherichia coli and the gene for a selectable marker, so that the possibility of recovering supF mutants containing gross rearrangements is low. The frequency of supF mutants obtained when untreated plasmid replicated in 293 cells was 1.4 X 10(-4). The frequency with BPDE-treated plasmid increased linearly as a function of the number of adducts, with 16 adducts per plasmid giving 38 X 10(-4). Polyacrylamide gel and agarose gel electrophoresis analysis of 137 plasmids with mutations in the supF gene indicated that 70% (21/30) from untreated plasmids contained deletions or insertions or showed altered gel mobility, whereas only 28% (30/107) of those derived from BPDE-treated plasmids contained such alterations. Of the 86 unequivocally independent mutants derived from BPDE-treated plasmids that were analyzed by sequencing, the majority (60/86) exhibited base substitutions. Mutants exhibiting frameshifts (insertions or deletions of one, two, or four base pairs) were also found, but they were a minority (11/86). In the progeny of BPDE-treated plasmids 61/71 base substitutions observed were transversions, with 45/61 G X C----T X A. Examination of the location of BPDE-induced mutations among the 85 base pairs in the structure of the tRNA revealed that 30% of the base substitutions occurred at two sites and 44% of the rest occurred at five other hot spots. Only 20% of all these base changes involved a site in which a guanine containing a BPDE adduct is predicted to be labile--i.e., a guanine that has a pyrimidine to its 5' side.     

Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells.

The role of UV radiation-induced photoproducts in initiating base substitution mutations in human cells was examined by measuring photoproduct frequency distributions and mutations in a supF tRNA gene on a shuttle vector plasmid transfected into DNA repair-deficient cells (xeroderma pigmentosum, complementation group A) and into normal cells. Frequencies of cyclobutane dimers and pyrimidine-pyrimidone (6-4) photoproducts varied by as much as 80-fold at different dipyrimidine sites within the gene. All transition mutations occurred at dipyrimidine sites, predominantly at cytosine, with a 17-fold variation in mutation frequency between different sites. Removal of greater than 99% of the cyclobutane dimers by in vitro photoreactivation before transfection reduced the mutation frequency while preserving the mutation distribution, indicating that (i) cytosine-containing cyclobutane dimers were the major mutagenic lesions at these sites and (ii) cytosine-containing non-cyclobutane dimer photoproducts were also mutagenic lesions. However, at individual dipyrimidine sites neither the frequency of cyclobutane dimers nor the frequency of pyrimidine-pyrimidone (6-4) photoproducts correlated with the mutation frequency, even in the absence of excision repair. Mutation hot spots occurred at sites with low or high frequency of photoproduct formation and mutation cold spots occurred at sites with many photoproducts. These results suggest that although photoproducts are required for UV mutagenesis, the prominence of most mutation hot spots and cold spots is primarily determined by DNA structural features rather than by the frequency of DNA photoproducts.     

Specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells in vivo.

The specific action of T4 endonuclease V on damaged DNA in xeroderma pigmentosum cells was examined using an in vivo assay system with hemagglutinating virus of Japan (Sendai virus) inactivated by UV light. A clear dose response was observed between the level of UV-induce unscheduled DNA synthesis of xeroderma pigmentosum cells and the amount of T4 endonuclease V activity added. The T4 enzyme was unstable in human cells, and its half-life was 3 hr. Fractions derived from an extract of Escherichia coli infected with T4V1, a mutant defective in the endonuclease V gene, showed no ability to restore the UV-induced unscheduled DNA synthesis of xeroderma pigmentosum cells. However, fractions derived from an extract of T4D-infected E. coli with endonuclease V activity were effective. The T4 enzyme was effective in xeroderma pigmentosum cells on DNA damaged by UV light but not in cells damaged by 4-nitroquinoline 1-oxide. The results of these experiments show that the T4 enzyme has a specific action on human cell DNA in vivo. Treatment with the T4 enzyme increased the survival of group A xeroderma pigmentosum cells after UV irradiation.     

Xeroderma pigmentosum variant cells are less likely than normal cells to incorporate dAMP opposite photoproducts during replication of UV-irradiated plasmids.

Xeroderma pigmentosum (XP) variant patients show the clinical characteristics of the disease, with increased frequencies of skin cancer, but their cells have a normal, or nearly normal, rate of nucleotide excision repair of UV-induced DNA damage and are only slightly more sensitive than normal cells to the cytotoxic effect of UV radiation. However, they are significantly more sensitive to its mutagenic effect. To examine the mechanisms responsible for this hypermutability, we transfected an XP variant cell line with a UV-irradiated (at 254 nm) shuttle vector carrying the supF gene as a target for mutations, allowed replication of the plasmid, determined the frequency and spectrum of mutations induced, and compared the results with those obtained previously when irradiated plasmids carrying the same target gene replicated in a normal cell line [Bredberg, A., Kraemer, K. H. & Seidman, M. M. (1986) Proc. Natl. Acad. Sci. USA 83, 8273-8277]. The frequency of mutants increased linearly with dose, but with a slope 5 times steeper than that seen with normal cells. Sequence analysis of the supF gene showed that 52 of 53 independent mutants generated in the XP variant cells contained base substitutions, with 62 of 64 of the substitutions involving a dipyrimidine. Twenty-eight percent of the mutations involved A.T base pairs, with the majority found at position 136, the middle of a run of three A.T base pairs. (In the normal cells, this value was only 11%.) If the rate of excision of lesions from supF in the two cell lines is equal, our data suggest that XP variant cells are less likely than normal cells to incorporate dAMP opposite bases involved in photo-products. If such incorporation also occurs during replication of chromosomal DNA, this could account for the hypermutability of XP variant cells with UV irradiation.     

Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA.

A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, 1053-1062]. This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line. Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form. Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a greater than 10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E. coli as part of a plasmid. The complete DNA sequences of three mutant genes have been determined. Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids.     

Identification and order of sequential mutations in beta-actin genes isolated from increasingly tumorigenic human fibroblast strains.

We have sequenced the mutant beta-actin gene of a tumorigenic human fibroblast cell line (HuT-14T) and found that it carries three mutations that alter the amino acids at positions 36, 83, and 244 as well as a 22-base-pair "insertion" sequence, in the 5' intron, not present in a wild-type gene. The less tumorigenic cell line HuT-14, a progenitor of HuT-14T, has the same codon-244 mutation and the insertion sequence but not the other two mutations. A nontumorigenic cell line that is related to HuT-14 but that has no beta-actin mutations does carry the intron-length polymorphism. We conclude that the mutation at codon 244 occurred first in a beta-actin allele already bearing the 22-base-pair intron insert and that mutations at codons 36 and 83 arose subsequently during the selection for the HuT-14T phenotype. Rat-2 cells synthesize the appropriate charge-variant species of mutant actin protein when transfected with either the singly or the triply mutated beta-actin gene.