Mutational hotspot variability in an ultraviolet-treated shuttle vector plasmid propagated in xeroderma pigmentosum and normal human lymphoblasts and fibroblasts

The mutagenesis shuttle vector, pZ189, was treated with ultraviolet (u.v.) radiation in vitro and passed through a DNA repair-deficient lymphoblastoid cell line derived from a patient with xeroderma pigmentosum complementation group A (XP-A) (XP12BE(EBV)) and a DNA repair-proficient lymphoblastoid cell line (GM606(EBV)). After u.v. treatment, plasmid survival was lower and mutation frequency higher with the XP-A cells mirroring the survival and mutagenesis of the host cells. The nature of the mutations in the suppressor tRNA marker gene was determined by direct sequence analysis. The G.C to A.T transition was the dominant (85%) base substitution mutation with the XP lymphoblasts and was the major (56%) base substitution mutation with the repair-proficient lymphoblasts. We found a G.C to A.T transition mutational hotspot with the XP lymphoblasts not seen in our previous experiments with fibroblasts from the same patient. Comparison of the data presented here with our results with DNA repair-deficient and DNA repair-proficient fibroblasts suggests that hotspot variability is not due to genetic polymorphism or repair capacity of the cells. Instead it appears that cellular factors can influence the probability of mutagenesis of modified DNA at particular sites.     

UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells.

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5'-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.     

Ultraviolet mutagenesis in human lymphocytes: The effect of cellular transformation

We have assessed the role of cellular transformation in ultraviolet (uv)-induced mutagenic events in human cells. To maintain uniformity of genetic background and to eliminate the effect of DNA repair, primary nontransformed lymphocytes (T-cells) and Epstein-Barr virus-transformed lymphocytes (B-cells) from one patient (XP12Be) with the DNA repair-deficient disorder xeroderma pigmentosum (group A) were transfected with the mutagenesis shuttle vector pZ189. Parallel control experiments were performed with primary, nontransformed lymphocytes from a normal individual and with a repair-proficient Epstein-Barr virus-transformed lymphocyte line (KR6058). pZ189 was treated with uv and introduced into the four cell lines by electroporation. Plasmid survival and mutations inactivating the marker supF suppressor tRNA gene in the recovered pZ189 were scored by transforming an indicator strain of Escherichia coli. Plasmid survival was reduced and mutation frequency elevated equally with both XP-A cell lines compared to both normal cell lines. Base sequence analysis of more than 250 independent plasmids showed that while the G:C----A:T base substitution mutation was found in at least 60% of plasmids with single or tandem mutations with all four cell lines, the frequency with the transformed XP-A (93%) cells was significantly higher (P less than 0.01) than that with the nontransformed XP-A cells (77%). In addition, with the transformed XP-A cells, there were significantly fewer plasmids with transversions and with mutations at a transversion hotspot (base pair 134) than with plasmids recovered from nontransformed XP-A cells. Interleukin-2 and phytohemagglutinin (used to maintain growth of the nontransformed lymphocytes) treatment of transformed XP12Be cells did not change overall plasmid survival or mutation frequency, but increased the transversion frequency and induced a mutational hotspot (at base pair 159), while another mutational hotspot (at base pair 123) disappeared. Thus we have demonstrated that in repair-deficient human cells, cellular transformation, while not affecting overall postuv plasmid survival and mutation frequency, does increase the susceptibility to G:C----A:T transition mutations, a type of mutation associated with uv-induced neoplasia.     

Mutagenesis of a shuttle vector plasmid in mammalian cells.

Recently we and others have reported a high frequency of mutagenesis of shuttle vector plasmids after passage in mammalian cells (Razzaque et al., Proc. Natl. Acad. Sci. U.S.A. 80:3010-3014, 1983; Calos et al., Proc. Natl. Acad. Sci. U.S.A. 80:3015-3019, 1983). The mutation frequency was determined by monitoring the integrity of a bacterial marker gene on the plasmid by standard microbiological procedures. Mutant plasmids contained deletions, insertions of cell DNA, and point mutations. The observed mutation frequency of 1% is much higher than that of cellular markers and could be due to the induction of a mutagenic environment by infection with a replicating plasmid. Alternatively, the hypermutagenesis may be due to some critical transient or persistent difference between the DNA in the plasmid and the cellular chromosome. We performed a number of experiments designed to distinguish between these alternatives, with particular reference to deletion mutagenesis. We conclude that mutagenesis was specific to the plasmid and propose that the majority of the deletion and insertion mutants were generated very early in the infection, before replication of the vector. However, some deletion mutagenesis also occurred after plasmid replication had begun.     

Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells.

A simian virus 40-based shuttle vector was used to characterize UV-induced mutations generated in mammalian cells. The small size and placement of the mutagenesis marker (the supF suppressor tRNA gene from Escherichia coli) within the vector substantially reduced the frequency of spontaneous mutations normally observed after transfection of mammalian cells with plasmid DNA; hence, UV-induced mutations were easily identified above the spontaneous background. UV-induced mutations characterized by DNA sequencing were found primarily to be base substitutions; about 56% of these were single-base changes, and 17% were tandem double-base changes. About 24% of the UV-induced mutants carried multiple mutations clustered within the 160-base-pair region sequenced. The majority (61%) of base changes were the G . C----A . T transitions; the other transition (A . T----G . C) and all four transversions occurred at about equal frequencies. Hot spots for UV mutagenesis did not correspond to hot spots for UV-induced photoproduct formation (determined by a DNA synthesis arrest assay); in particular, sites of TT dimers were underrepresented among the UV-induced mutations. These observations suggest to us that the DNA polymerase(s) responsible for mutation induction exhibits a localized loss of fidelity in DNA synthesis on UV-damaged templates such that it synthesizes past UV photoproducts, preferentially inserting adenine, and sometimes misincorporates bases at undamaged sites nearby.     

A shuttle vector plasmid for studying carcinogen-induced point mutations in mammalian cells

We have constructed a shuttle vector plasmid for studying mutagenesis in mammalian cells. The plasmid replicates in cell lines permissive for SV40 virus as well as in the bacterium Escherichia coli and carries a bacterial suppressor tRNA gene (supF) that can serve as a mutagenesis marker. The plasmid replicates as efficiently as SV40 virus in African Green Monkey kidney CV1 cells, indicating that all traces of the inhibitory sequences normally found in pBR322 and its derivatives have been removed. The design of the plasmid and the small size of the mutagenesis target gene decrease the probability of recovering spontaneous deletion mutations that have been shown to occur at high frequency during passage in mammalian cells. The frequency of spontaneous-mutant plasmids recovered after passage in CV1 cells is substantially lower than with other vectors described previously. When the plasmid DNA is treated with UV radiation before passage in CV1 cells, mutants are observed at a frequency about 20-fold above the spontaneous background.     

Suppression of UV mutagenicity by human interferon

Effects of human interferon (HuIFN)-alpha on UV mutagenicity were examined in a human cell strain, RSa, and xeroderma pigmentosum (XP)-derived fibroblasts (XP1KY). The frequency of ouabain-resistance mutation in UV-irradiated RSa cells was unusually high (Suzuki et al., 1985), but that in cells pretreated with HuIFN-alpha before irradiation was reduced. 6-Thioguanine-resistance mutation was also depressed in XP1KY cells treated with HuIFN-alpha before irradiation. However, the depression of UV mutagenicity by HuIFN-alpha was lessened by treatment with cycloheximide immediately after UV irradiation. The relationship between HuIFN-depressed UV mutagenicity and HuIFN-affected DNA-repair and repair-related functions is discussed.     

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.     

Mutation spectrum in gamma-irradiated shuttle vector replicated in ataxia-telangiectasia lymphoblasts.

Cells from ataxia-telangiectasia (AT) patients are hypersensitive to the lethal effects of ionizing radiation. To assess radiation mutagenesis in these cells, the SV40-based shuttle vector, pZ189, was used to analyze gamma-ray-induced mutations following the plasmid's replication in AT lymphoblasts. Progenies from the AT line GM2783 exposed to 50 Gy showed a mutation frequency of 7.6 x 10(-3), 63-fold over background; surviving plasmids were 3.4% of control. Both values were essentially the same as those of irradiated plasmids replicated in a normal lymphoblast line, GM606. In addition, pZ189 exposed to 25 Gy of gamma radiation and replicated in another normal lymphoblast line and in cells of two additional AT lymphoblast lines showed similar mutation frequencies and percentages of surviving plasmids. Qualitative comparison of plasmid mutations from AT and normal cells showed no significant differences, indicating that the damaged DNA was repaired with similar fidelity in AT and normal cells. These studies suggest that there is no correlation between the enhanced sensitivity of AT cells to killing by ionizing radiation and gamma-radiation-induced mutagenesis of plasmid DNA processed in these cells.     

High sensitivity but normal DNA-repair activity after UV irradiation in Epstein--Barr virus-transformed lymphoblastoid cell lines from Chediak--Higashi syndrome

We established lymphoblastoid cell lines from 2 children with Chediak--Higashi syndrome (CHS), 2 xeroderma pigmentosum (XP) patients and control donors after transformation of peripheral lymphocytes by Epstein--Barr virus (EBV). We used these lymphoblastoid cell lines to investigate repair activity after ultraviolet irradiation. Cell survival of both CHS lymphoblastoid cell lines after irradiation by UV and treatment by 4-nitroquinoline 1-oxide (4NQO) fell between those of the XP and control cell lines. Unscheduled DNA synthesis of CHS cells after UV irradiation occurred at rates similar to those of control cells.     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. II. Sequence analysis of alpha-particle-induced point mutations

The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells. Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E. coli. The mutations were characterized by sequencing the tRNA gene. The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%). The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs. The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences. For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA. Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA. The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different.     

Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light.

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded.     

Cell cycle-dependent strand bias for UV-induced mutations in the transcribed strand of excision repair-proficient human fibroblasts but not in repair-deficient cells.

To study the effect of nucleotide excision repair on the spectrum of mutations induced in diploid human fibroblasts by UV light (wavelength, 254 nm), we synchronized repair-proficient cells and irradiated them when the HPRT gene was about to be replicated (early S phase) so that there would be no time for repair in that gene before replication, or in G1 phase 6 h prior to S, and determined the kinds and location of mutations in that gene. As a control, we also compared the spectra of mutations induced in synchronized populations of xeroderma pigmentosum cells (XP12BE cells, which are unable to excise UV-induced DNA damage). Among the 84 mutants sequenced, base substitutions predominated. Of the XP mutants from S or G1 and the repair-proficient mutants from S, approximately 62% were G.C----A.T. In the repair-proficient mutants from G1, 47% were. In mutants from the repair-proficient cells irradiated in S, 71% (10 of 14) of the premutagenic lesions were located in the transcribed strand; with mutants from such cells irradiated in G1, only 20% (3 of 15) were. In contrast, there was no statistically significant difference in the fraction of premutagenic lesions located in the transcribed strand of the XP12BE cells; approximately 75% (24 of 32) of the premutagenic lesions were located in that strand, i.e., 15 of 19 (79%) in the S-phase cells and 9 of 13 (69%) in the G1-phase cells. The switch in strand bias supports preferential nucleotide excision repair of UV-induced damage in the transcribed strand of the HPRT gene.     

Induced mutagenesis of plasmid and chromosomal genes inserted into plasmid DNA. I. The mutagenic action of radiation

This paper describes the results of treating plasmid DNA in vitro with mutagens, to obtain mutations both in plasmid genes and chromosomal genes comprised within the plasmid, thus avoiding disorganization characteristic of in vivo mutagenesis. The model system is represented by DNA of RSF2124 responsible for colicine E1 synthesis and resistance to ampicillin. Col- mutants were looked for after exposure to UV- and gamma-irradiation. The lethal effect was estimated as inactivation of the ampicillin resistance marker. After reisolation from mutant transformant of the plasmid DNA, the novel character and resistance to ampicillin proved to retain in the course of subsequent transformations and passages of transformed colonies, suggesting the mutational nature of the changes. Exposure of RSF2124 to short-wave UV-irradiation (lambda = 254 nm) produced a pronounced mutagenic effect: the relative quantity of Col- mutants under optimal conditions of mutagenesis increased about 10 times. In the case of W-reactivation (additional UV-irradiation of C600 wild type cells) of lethal lesions, a 95% reliable increase in mutagenic effect was observed. Significant enhancement of mutagenesis (about 4-fold) was detected when only recipient cells were exposed to low doses of UV (the so-called indirect UV mutagenesis). Thus, with regard to W- and indirect UV mutagenesis, the plasmid DNA behaves like DNA of temperate phages which suggests their evolutionary relationship. Treatment of plasmid DNA with acridine orange prior to UV, only protected from lethal lesions. Gamma-irradiation (60Co) at the dose producing 100-fold inactivation, increased the yield of Col- mutants by one order of magnitude. The presence of RSF2124 plasmid in a cell does not affect its UV sensitivity.     

DNA photolyase in E. coli: effects on UV mutagenesis by plasmids expressing the phr gene

Derivatives of an E. coli plasmid pKY33 are described having specific insertions or deletions that effect or do not effect the phr gene (for DNA photolyase) carried in this plasmid. The various plasmids are tested to determine which cause an inhibition of UV mutagenesis producing glutamine tRNA ochre suppressor mutations. The inhibition is found to require a functional phr gene, which substantiates our earlier report that amplified DNA photolyase interferes specifically with a category of mutagenesis involving targeting by a pyrimidine dimer.     

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.     

Abnormal processing of transfected plasmid DNA in cells from patients with ataxia telangiectasia.

In order to assess spontaneous mutability and accuracy of DNA joining in ataxia telangiectasia, a disorder with spontaneous chromosome breakage, the replicating shuttle vector plasmid, pZ189, was transfected into SV40 virus-transformed fibroblasts from ataxia telangiectasia patients. The ataxia telangiectasia fibroblasts showed elevated frequency of micronuclei, a measure of chromosome breakage. The spontaneous mutation frequency was normal with circular plasmids passed through the ataxia telangiectasia line. These results were compared to those with transformed fibroblasts from a patient with xeroderma pigmentosum, and from a normal donor. Mutation analysis revealed spontaneous point mutations and deletions in the plasmids with all 3 cell lines, however, insertions or complex mutations were only detectable with the ataxia telangiectasia line. To assess DNA-joining ability, linear plasmids which require joining of the DNA ends by host cell enzymes for survival, were transfected into the cells. We found a 2.4-fold less efficient DNA joining in ataxia telangiectasia fibroblasts (p = 0.04) and a 2.0-fold higher mutation frequency (p less than 0.01) in the recircularized plasmids than with the normal line. Plasmid DNA joining and mutation frequency were normal with the xeroderma pigmentosum fibroblasts. These findings with the ataxia telangiectasia fibroblasts of abnormal types of spontaneous mutations in the transfected plasmid and inefficient, error-prone DNA joining may be related to the increased chromosome breakage in these cells. In contrast, an EB virus-transformed ataxia telangiectasia lymphoblast line with normal frequency of micronuclei showed normal types of spontaneous mutations in the transfected plasmid and normal frequency of DNA joining which was error-prone. These data indicate that mechanisms that produce chromosome breakage in ataxia telangiectasia cells can be reflected in processing of plasmid vectors.     

Mutation spectra induced by alpha-acetoxytamoxifen-DNA adducts in human DNA repair proficient and deficient (xeroderma pigmentosum complementation group A) cells

Tamoxifen, a breast cancer drug, has recently been approved for the chemoprevention of this disease. However, tamoxifen causes hepatic carcinomas in rats through a genotoxic mechanism and increases the risk of endometrial tumors in women. DNA adducts have been detected at low levels in human endometrium, and there is much interest in determining whether DNA damage plays a role in tamoxifen-induced endometrial carcinogenesis. This study investigates the mutagenicity of tamoxifen DNA adducts formed by alpha-acetoxytamoxifen, a reactive ester producing the major DNA adduct formed in livers of tamoxifen-treated rats. pSP189 plasmid DNA containing the supF gene was treated with alpha-acetoxytamoxifen and adduct levels (0.5-8.0 adducts per plasmid) determined by (32)P-postlabeling. Adducted plasmids were transfected into nucleotide excision repair proficient (GM00637) or deficient (GM04429, XPA) human fibroblasts. After replication, plasmids were recovered and screened in indicator bacteria. Relative mutation frequencies increased with the adduct level, with 1.3-3.6-fold higher numbers of mutations in the XP cells compared to the GM00637 cells, indicating that NER plays a significant role in the removal of these particular tamoxifen DNA adducts. The majority of sequence alterations (91-96%) occurred at GC base pairs, as did mutation hotspots, although the type and position of mutations was cell-specific. In both cell lines, as the adduct level increased, the proportion of GC --> AT transitions decreased and GC --> TA transversions, mutations known to arise from the major tamoxifen adducts, increased. Given the high mutagenicity of dG-N(2)-tamoxifen adducts, if not excised, they may potentially contribute to the initiation of endometrial cancer in women.     

The Role of Pyrimidine Dimers as Premutagenic Lesions: A Study of Targeted VS. Untargeted Mutagenesis in the lacI Gene of ESCHERICHIA COLI

We have employed conjugal transfer of an F' lac episome to examine targeted and untargeted mutagenesis in the lacI gene of Escherichia coli and to determine the relative importance of pyrimidine dimers as premutational UV lesions compared to (6-4) photoproducts that also may have a mutational role. This conjugal system allowed us to assess the premutagenic role of UV lesions independently from any role as inducers of SOS functions. F' DNA was transferred to an SOS-induced recipient strain from: unirradiated donor cells, UV-treated donor cells or donor cells that were irradiated and then exposed to photoreactivating light. The results indicate that SOS-related, untargeted events may account for as much as one-third of the nonsense mutations (i.e., base substitutions) recovered after undamaged F' DNA is transferred to UV-irradiated recipients. When the donor strain also is irradiated, in excess of 90% of the mutations detected following conjugation appear to be targeted. Photoreactivation of the UV-treated donors cells, prior to F' transfer to the SOS-induced recipient strain, demonstrated that in this experimental system virtually all recovered UV-induced mutations are targeted by photoreactivable lesions. We presume that these lesions are pyrimidine dimers because (6-4) photoproducts are not photoreactivable.