INT . J . RADIAT . BIOL .,
1990,
VOL .
57, NO . 4, 665-676
Mechanisms and consequences of mutation induction in mammalian cells* A . SARASIN, F . BOURRE, L . DAYA-GROSJEAN, A . GENTIL, C . MADZAK and A . STARY
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Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, B .P . n° 8, 94802 Villejuif Cedex, France Mutations have been studied for several decades in order to understand biological processes of great significance and the selection of better-adapted species . Our knowledge both of mutation spectra induced by genotoxic agents and the mechanisms involved in DNA damage processing is more advanced in bacteria than in animal cells . However, the use of new technologies such as shuttle vectors or the polymerase chain reaction will undoubtedly allow rapid progress in the next few years . Shuttle vectors consist of target sequences for monitoring mutagenic activity and additional sequences permitting DNA replication and selection, both in bacteria and in mammalian cells . These plasmids are very efficient in allowing the production of mutation spectra of a particular genotoxin in animal cells . In most cases, base substitutions occur predominantly at the sites of base damage and the type of substitution depends on the kind of damage . This has been well characterized using ultraviolet (UV) light as a mutagen . UV-induced mutations are targeted opposite pyrimidine-pyrimidine sites, where the two major UV lesions are produced . The direct relationships existing between mutation and cancer are exemplified by some hereditary diseases where deficiency in an enzymatic repair system is linked to a high incidence of tumours . Similarly, activation of some cellular proto-oncogenes occurs via specific point mutations . A correlation does exist between the mutation spectra found in model systems and the specific mutation found in the activated oncogene in tumours induced by a given genotoxin . This is particularly well illustrated in the DNA repair deficiency syndrome, xeroderma pigmentosum . The specific mutations found in activated ras oncogenes isolated from UV-stimulated skin tumours correlate well with the mutagenic properties of unrepaired UV-induced DNA lesions . 1.
Introduction Mutations occur in all living systems at various rates depending on the cellular types and the genetic alterations considered . Background mutations are due to unknown causes while induced mutations can be observed after treatment with a genotoxic agent . Most mutations exhibit deleterious effects on the cell or on individuals, but specific genetic changes appear to play a positive role in some basic processes such as species evolution or immunoglobulin gene variability . Numerous types of genetic alterations ranging from large chromosomal changes to a single nucleotide modification have been associated with human diseases such as chromosome gain in Down's syndrome, large deletions in the hereditary retinoblastoma or point mutation in sickle cell anemia . It is clear that a better understanding of the mechanisms of mutation induction will allow a better knowledge of disease appearance leading to more efficient prevention . For obvious *Presented at the 22nd Annual Meeting of the European Society for Radiation Biology, 12-16 September 1989, Brussels . 0020-7616/90 $3 .00 © 1990 Taylor & Francis Ltd
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reasons, point mutations or mutations involving a few bases have been studied in more detail than large genomic rearrangements . Owing to the availability of numerous mutants, the various pathways leading to mutagenesis have been elucidated at the molecular level in E . coli. The absence of well defined mutants in mammalian cells makes it more difficult to study mutagenesis . Moreover, the structure and organization of the mammalian chromatin are so complex that the fate of a given DNA lesion can vary as a function of the cell cycle, the chromosome structure, the transcriptional activity of the gene or the nucleotide sequence . Nevertheless, the various types of DNA lesion processing have been established as shown in figure 1 . In particular, it has been elegantly demonstrated by Hanawalt's group that UV-induced DNA lesions are preferentially repaired in transcriptionally active genes and more on the transcribed strand than on the non-transcribed one (Hanawalt 1987) (figure 1) . Moreover, the mutagenic efficiency of a given lesion will also vary widely as a function of its location in the chromatin structure . Recent advances in technology such as the polymerase chain reaction (PCR) or the use of exogenous probes (animal virus or shuttle vectors) have, however, a
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6 Figure 1 . Various types of DNA lesion processing . DNA lesions are induced on doublestranded DNA due to either `spontaneous' events or genotoxic agents (1) . A complete repair gives rise to the original genetic message (2) while in the absence of any successful repair or tolerance mechanisms, DNA lesions can lead to cellular death (7) . Most DNA lesions represent a block to RNA transcription (3) and DNA replication (5) . Preferential repair has been shown to occur on the transcribed strand (a*) of active genes (4) . DNA lesions on the leading strand constitute a block to replication (5b) while on the lagging strand (5b*) fork displacement may continue . It is believed that trans-lesion synthesis may induce mutation targeted opposite DNA lesions (6) .
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facilitated the determination of mutation spectra in specific genes . PCR is an in vitro method of enzymatic DNA amplification for producing large quantities of a given DNA fragment (Bos et al . 1986) . Hence, PCR followed by DNA sequencing allows a rapid assay for characterizing mutations in DNA isolated from a very small number of mutant cells . This technique has already been used to define spontaneous or induced mutation spectra in integrated DNA sequences (Tindall and Stankowski 1989) . It is obvious that this new technique will be more widely used, and that numerous mutation spectra in a variety of genes will be rapidly obtained . The role of mutations in carcinogenesis has been evoked over several decades, but it is mainly due to the analysis of oncogene activation that a relationship between point mutation and cancer initiation has been demonstrated . Studies on human hereditary diseases such as xeroderma pigmentosum, in which DNA repair deficiency is directly responsible for cancer development, allow a better understanding of the link between DNA lesions, mutation and cancer induction .
2.
Animal virus and transient SV40-shuttle vectors as probes The complex organization of the genome of mammalian cells has necessitated the use of exogenous probes to analyse mutagenesis at the molecular level, The majority of these studies have been carried out with the help of two kinds of probes : animal viruses and shuttle vectors . Among the former, Simian virus 40 (SV40) (Sarasin and Benoit 1980, 1986), Herpes simplex virus (Das Gupta and Summers 1978), adenovirus (Day and Ziolkowski 1981) and parvovirus (Cornelis et al . 1982) have been frequently used as molecular probes to study the effects of physical or chemical DNA-damaging agents . Many experiments have been carried out with SV40 in which permissive simian cells were infected with a temperature-sensitive mutant of SV40 pre-treated in vitro with genotoxic agents . The reversion toward a wild-type phenotype of growth was the mutagenic criterion . The mutants were selected and plaque purified, mutation spectra being established by DNA sequencing (Bourre and Sarasin 1983, Gentil et al. 1986) . More recently, vectors able to replicate in mammalian cells as well as in bacteria were developed . These shuttle vectors are grown in mammalian cells and subsequently screened for mutation in bacteria . Numerous shuttle vectors have been constructed and most of them are SV40-based . Typically, as shown in figure 2, a shuttle vector carries two origins of replication : a mammalian one derived from a eukaryotic virus (EBV, SV40), allowing replication of the vector in mammalian cells, and a plasmidic one, allowing replication in bacteria . Plasmid DNA is treated in vitro with DNA-damaging agents prior to transfection in mammalian cells . After its replication the plasmid DNA is harvested from the cells where the mutations occur and screened in bacteria . Mutants are selected in the target gene (sup F, lac I etc) and the mutation spectra are determined by DNA sequencing (Calos et al. 1983, Razzaque et al. 1983) . The two biological models present some advantages and disadvantages . Indeed, transient shuttle vectors allow analysis at the molecular level of a great number of mutants selected in a forward assay, whereas with the animal viruses only a small number of mutants are analysed by a time-consuming reversion assay . One of the main disadvantages in the use of shuttle vectors is the requirement of DNA transfection to introduce the mutational target into the cells, while the animal viruses are introduced into host cells by natural infection . When using DNA transfection only 5-10 per cent of the cells effectively take up the DNA, and it is not
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MAMMALIAN
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ORI
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RESISTANCE BACTERIAL ORI
Figure 2 . Schematic representation of a shuttle vector genetic map . Mammalian ORI (SV40, Py or EBV derived) is a DNA sequence from animal virus replication origin allowing replication in mammalian cells by using a viral Trans acting protein (SV40 TAg, Py TAg or EBNA1 protein) . Bacterial ORI is a DNA sequence from the replication origin of the pBR plasmid . Selection gene (resistance to G418 or hygromycin) and Antibiotic resistance gene (Amp, Tet, Cm or Neo) code for proteins allowing selection of the plasmids respectively in mammalian cells and in bacteria . Target gene (gal K, lac I, lac Z, sup F, lac 0, HSV-tk, gpt or aprt) represents sequence used for screening mutations .
known whether these cells represent either a random part of the cell population or a subpopulation in a different physiological state . Moreover, this step may be responsible for the higher background mutation frequency observed with transient shuttle vectors compared with that measured with animal viruses or endogenous genes (for review see Bourre et al . 1989b) . To avoid transfection problems Yates et al. (1984) reported on the use of Epstein-Barr virus episomal shuttle vectors . These plasmids are maintained stably in human cells and can be rescued in bacteria to screen mutagenesis . Nevertheless, they are technically difficult to work with, and it is not possible to treat them independently of the cells . 3 . Origins of mutation 3 .1 Spontaneous mutation Spanning the range from bacteria to higher eukaryotes, the mutation rate is remarkably low, between 10 -9 and 10 -12 errors per base pair per generation (Cox 1976) . This low spontaneous mutation frequency could result from various processes . In vitro studies show that the accuracy of eukaryotic DNA polymerases depends on the kind of polymerase, the type of 'mispair' that is formed and the surrounding
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sequence (Lai and Beattie 1988) . Error rates measured in in vitro replication systems remain much higher than for chromosomal loci in vivo (Roberts and Kunkel 1988), suggesting that additional fidelity factors, such as the proofreading activity, may be active during chromosomal replication . Moreover, mismatch repair appears to be very efficient in vivo on newly synthesized DNA . Despite these cellular mechanisms, spontaneous mutations appear during DNA replication . They could also be due to spontaneous chemical modifications of the DNA . In particular it has been shown that 10,000 depurinations per day occur in a mammalian cell (Lindahl 1982) . They are due to the spontaneous hydrolysis of the N-glycosilic bond or appear as intermediates in DNA repair . Apurinic sites (Ap) are recognized and repaired by specific Ap-endonucleases, ubiquitous in both prokaryotes and eukaryotes (Friedberg 1985) . This might indicate the high toxicity of unrepaired Ap sites . Indeed, it has been shown in mammalian cells that the presence of Ap sites strongly decreases the survival of treated SV40 virus, -and that they are highly mutagenic (Gentil et al . 1984) . Another possible cause for background mutations is the spontaneous de-amination of cytosine, especially of 5-methyl cytosine into thymine (Bird 1980) . 5-mC is found in the dinucleotide CpG which is the major methylated sequence in animals . The implication of mCpG in spontaneous mutagenesis is supported by studies comparing the sequences of 5 S RNAs of Xenopus laevis (Brownlee et al . 1972) or of the chicken ovalbumin gene (Mandel and Chambon 1979) . Among the factors favouring the occurrence of mutations it has been proposed that secondary structures could play a role . Indeed, in E . coli, frameshifts occur frequently at reiterated base sequences (Streisinger and Owen 1985) and addition/deletion events concern mostly repeated units (Schaaper et al . 1986) suggesting a misaligned pre-mutational intermediate containing an (some) extrahelical nucleotide(s) . Similarly, deletions account for 10 per cent of the spontaneous mutants at the aprt locus of hamster cells, and occur by non-homologous recombination between short sequence repeats (Nalbantoglu et al . 1986) . Ripley has proposed that the in vivo processing of misaligned quasipalindromic DNA sequences could produce frameshift and base substitution mutations . Analyses of spontaneous frameshifts in E . coli (De Boer and Ripley 1984) and in Saccharomyces cerevisiae (Ripley 1982) correlate with this hypothesis . Finally, it has been proposed that transposable genetic elements would be responsible for numerous mutations, although the mechanisms by which they occur are not known (for review see Sankaranarayanan, 1986) .
3 .2
Induced mutation It is now well known that DNA-damaging agents induce mutations . Therefore it is important to define the location of the induced mutation in relation to the lesion produced, which permits one to estimate which lesions are pre-mutagenic . Among DNA damaging agents, 254nm ultraviolet (UV) light in particular has been extensively analysed, since it represents one of the major carcinogens in humans . UV-induced mutations in mammalian cells were found to occur almost exclusively at dipyrimidine sequences in studies with SV40 virus (Bourre and Sarasin 1983, Braggaar et al . 1985), shuttle vectors (Hauser et al . 1986, Hsia et al. 1989) or at endogenous loci (Drobetsky et al . 1987) . These sequences being potential UVinduced lesion sites, mutagenesis appears to be essentially targeted (figure 3) . The main lesions induced by UV-irradiation on DNA are the major cy-
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1000-fold elevated skin cancer incidence is due to the complete or partial absence of repair in UV-damaged DNA, particularly of pyrimidine dimers and pyrimidine (6-4) pyrimidones (figure 1) . We have analysed DNA from 12 epithelial tumours (basal or squamous cell carcinomas) obtained from young XP patients . DNA from two independent tumours from the same XP child were able to form foci in the 3T3 cells due to the transforming activity of the N-ras gene (Suarez et al. 1989) . Using the polymerase chain reaction for gene amplification and differential hybridization with synthetic oligonucleotide probes (Bos et al . 1986) we were able to determine the presence of a critical point mutation in codon 61 of the N-ras gene (Suarez et al . 1989) . This results in the substitution of a glutamine by a histidine residue which is sufficient to activate the Ras protein (figure 4) . Recently a tumour from another XP patient was found to contain an activated Ki-ras gene, due to a point mutation in codon 12 resulting in the substitution of a glycine by a valine (figure 4) . We interpret these results in the model depicted in figure 4 : UV-irradiation of the epithelial cells in the exposed areas of the skin induces lesions which if unrepaired could lead after replication to the incorporation of a wrong base opposite the pyrimidine involved in the lesion . Hence, the XP syndrome represents a human cancer model comparable to the animal tumour studies where oncogene activation by a point mutation is correlated with a specific type of adduct present on the cellular DNA . However, as most neoplasms develop with a long latent period and go through a series of progressive pathological stages, oncogenesis must involve more than a simple activation of a single transforming gene . For example, benign skin papilloma induced in animals or in humans contain activated ras genes (often Ha-ras) but the majority of these tumours regress and never give rise to cancers (Balmain et al. 1984, Corominas et al . 1989) . Secondary genetic events therefore seem necessary for tumour progression . Hence, we screened the XP tumours for the existence of other genetic modifications, particularly of other oncogenes . Indeed, the majority of the tumours exhibit amplification of the myc and/or Ha-ras oncogene . In particular, the Ha-ras gene was amplified and often rearranged in over 40 per cent of the tumours, a level significantly higher than seen (1 per cent) in human tumours in general . This high level of amplification in XP cells is probably due to UV-induced lesions blocking replication and resulting in multiple cycles of abortive reinitiation (Lavi 1981) . This type of random amplification in genes such as myc or Ha-ras
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Figure 4 . A model for proto-oncogene activation by point mutation in UV-induced epithelioma from xeroderma pigmentosum . Irradiation by UV light induces the formation of pyrimidine dimers or of pyrimidine(6-4)pyrimidone photoproducts (A) . In XP cells these lesions are not repaired and replication of the damaged DNA results in a point mutation opposite the lesion . When this occurs in a crucial codon of a protooncogene, such as codon 61 of the N-ras gene or codon 12 of the Ki-ras gene, the modification of the genetic code results in the activation of the gene (transforming activity) . Numbers correspond to the nucleotides in the coding sequence .
probably leads to selective cell proliferation which, together with activation of a second oncogene (e .g . N-ras), permits progression to a fully transformed phenotype . The results obtained from our study of XP tumours have substantiated the hypothesis made in animal studies as applicable to human carcinogenesis . A direct correlation is now possible between the mutation spectrum induced by a specific genetic lesion and the types of mutations observed in activated oncogenes . Acknowledgements This work was supported by grants from the Commission of the European Communities, Brussels, Belgium (n . B16-E-163 F), from the Ministere de la Recherche et de l'Enseignement Superieur and from the Association pour la Recherche sur le Cancer, Villejuif, France .
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References BALMAIN, A ., and BROWN, K ., 1988, Oncogene activation in chemical carcinogenesis . Advances in Cancer Research, 51, 147-182 . BALMAIN, A., RAMSDEN, M ., BOWDEN, G . T., and SMITH, J ., 1984, Activation of the mouse cellular Harvey-ras gene in chemically induced benign skin papilloma . Nature (London), 307, 658-660. BARBACID, M ., 1987, Ras genes . Annual Review of Biochemistry, 56, 779-827 . BIRD, A . P ., 1980, DNA methylation and the frequency of CpG in animal DNA . Nucleic Acids Research, 8, 1499-1503 . Bos, J . L ., VERLAAN-DE-VRIES, M ., MARSHALL, C . J ., VEENEMAN, G . H ., VAN Boom, J . H ., and VAN DER EB, A ., 1986, Human gastric carcinoma contains a single mutated and an amplified normal allele of the Ki-ras oncogene . Nucleic Acids Research, 14, 1209-1217 . BOURRE, F., and SARASIN, A ., 1983, Targeted mutagenesis of SV40 DNA induced by UV light . Nature (London), 305, 68-70 . BOURRE, F., BENOIT, A ., and SARASIN, A., 1989a, Respective role of pyrimidine dimer and pyrimidine(6-4)pyrimidone photoproducts in UV mutagenesis of SV40 DNA in mammalian cells . Journal of Virology, 63, 4520-4524 . BOURRE, F ., RENAULT, G ., AND GENTIL, A ., 1989b, From Simian Virus 40 to transient shuttle vectors in mutagenesis studies . Mutation Research, 220, 107-113 . BRAGGAAR, H ., CORNELIS, J . J ., VAN DER LUBBE, J . L . M ., and VAN DER EB, A . J ., 1985, Mutagenesis in UV-irradiated Simian virus 40 occurs predominantly at pyrimidine doublets . Mutation Research, 142, 75-81 . BRASH, D . E ., SEETHARAM, S ., KRAEMER, K . H ., SEIDMAN, M . M ., and BREDBERG, A ., 1987, Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells . Proceedings of the National Academy of Sciences, USA, 84, 3782-3786 . BROWNLEE, G . G ., CARTWRIGHT, E ., MCSHANE, T ., and WILLIAMSON, R ., 1972, The nucleotide sequence of somatic 5S RNA from Xenopus lcevis . FEBS Letters, 25, 8-12 . BURNOUF, D ., KOEHL, P ., and FUCHS, R. P . P ., 1989, Single adduct mutagenesis : strong effect of the position of a single acetylaminofluorene adduct within a mutation hot spot . Proceedings of the National Academy of Sciences, USA, 86, 4147-4151 . CALOS, M . P ., LEBKOWSKI, J . S ., and BOTCHAN, M . R ., 1983, High mutation frequency in DNA transfected into mammalian cells . Proceedings of the National Academy of Sciences, USA, 80, 3015-3019 . CLEAVER, J . E ., 1968 . Defective repair replication of DNA in xeroderma pigmentosum . Nature (London), 218, 652-656 . CORNELIS, J . J ., Su, Z . Z ., and ROMMELAERE, J ., 1982, Direct and indirect effects of UV-light on the mutagenesis of parvovirus H-1 . EMBO Journal, 1, 693-699 . COROMINAS, M ., KAMINO, H ., LEON, J ., and PELLICER, A ., 1989, Oncogene activation in human benign tumors of the skin (keratoacanthomas) : is ras involved in differentiation as well as proliferation? Proceedings of the National Academy of Sciences, USA, 86,6372-6376 . Cox, E . C ., 1976, Bacterial mutator genes and the control of spontaneous mutation . Annual Review of Genetics, 10, 135-156 . DAs GUPTA, U . B ., and SUMMERS, W . C ., 1978, Ultraviolet reactivation of Herpes simplex virus is mutagenic and inducible in mamalian cells . Proceedings of the National Academy of Sciences, USA, 75, 2378-2381 . DAY III, R . S ., and ZIOLKOWSKI, C . H . J ., 1981, UV-induced reversion of adenovirus 5 ts2 infecting human cells . Photochemistry and Photobiology, 34, 403-406 . DE BOER, J . G ., and RIPLEY, L . S ., 1984, Demonstration of the production of frameshift and base-substitution mutations by quasipalindrotnic DNA sequences . Proceedings of the National Academy of Sciences, USA, 81, 5528-5531 . DROBETSKY, E . A ., GROSOVSKY, A . J ., and GLICKMAN, B . W ., 1987, The specificity of UV induced mutations at an endogenous locus in mammalian cells . Proceedings of the National Academy of Sciences, USA, 84, 9103-9107 . FRIEDBERG, E . C ., 1985, DNA Repair (New York : Freeman) .
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FUCHS, R . P . P ., 1983, DNA binding spectrum of the carcinogen N-acetoxy-N-2acetylaminofluorene significantly differs from the mutation spectrum . Journal of Molecular Biology, 177,173-180 . FUCHS, R . P . P ., LEFEVRE, J . F ., POUYET, J ., and DAUNE, M . P ., 1976, Comparative orientation of the fluorene residue in native DNA modified by N-acetoxy-N-2acetylaminofluorene and two 7-halogeno derivatives . Biochemistry, 15, 3347-3351 . GENTIL, A ., MARGOT, A ., and SARASIN, A ., 1984, Apurinic sites cause mutations in simian virus 40 . Mutation Research, 129, 141-147 . GENTIL, A ., MARGOT, A ., and SARASIN, A ., 1986, 2-(N-acetoxy-N-acetylamino)fluorene mutagenesis in mammalian cells : sequence-specific hot spot. Proceedings of the National Academy of Sciences, USA, 83, 9556-9560 . HANAWALT, P . C ., 1987, Preferential DNA repair in expressed genes . Environmental Health Perspectives, 76, 9-14 . HANAWALT, P . C ., and SARASIN, A ., 1986, Cancer prone hereditary diseases with DNA processing abnormalities . Trends in Genetics, 2, 124-129 . HAUSER, J ., SEIDMAN, M . M ., SIDUR, K ., and DIXON, K ., 1986, Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells . Molecular and Cellular Biology, 6, 277-285 . HSIA, H . C ., LEBKOWSKI, J . S ., LEONG, P . M ., CALOS, M . P., and MILLER, J . H ., 1989, Comparison of ultraviolet irradiation-induced mutagenesis of the lacl gene in Escherichia coli and in human 293 cells . Journal of Molecular Biology, 205, 103-113 . KNUDSON, A . G ., 1986, Genetics of human cancer . Annual Review of Genetics, 20, 231-251 . KOEHL, P ., BURNOUF, D ., and FUCHS, R . P . P ., 1989, Construction of plasmids containing a unique acetylaminofluorene adduct located within a mutation hot spot . A new probe for frameshift mutagenesis . Journal of Molecular Biology, 207, 355-364 . LAI, M .-D ., and BEATTIE, K . L ., 1988, Influence of DNA sequence on the nature of mispairing during DNA synthesis . Biochemistry, 27, 1722-1728 . LAVI, S ., 1981, Carcinogen-mediated amplification of viral DNA sequences in simian virus 40-transformed Chinese hamster embryo cells . Proceedings of the National Academy of Sciences, USA, 78, 6144-6148 . LINDAHL, T ., 1982, DNA repair enzymes . Annual Review of Biochemistry, 51, 61-87 . MANDEL, J . L., and CHAMBON, P ., 1979, DNA methylation : organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes . Nucleic Acids Research, 7, 2081-2103 . NALBANTOGLU, J ., HARTLEY, D ., PHEAR, G ., TEAR, G ., and MEUTH, M ., 1986, Spontaneous deletion formation at the aprt locus of hamster cells : the presence of short sequence homologies and dyad symmetries at deletion termini . EMBO Journal, 5, 1199-1204 . PROTIC-SABLJIC, M ., TUTEJA, N ., MUNSON, P . J ., HAUSER, J ., KRAEMER, K . H ., and DIXON, K ., 1986, UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells. Molecular and Cellular Biology, 6, 3349-3356 . RAZZAQUE, A ., MIZUSAWA, H ., and SEIDMAN, M . M ., 1983, Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells . Proceedings of the National Academy of Sciences, USA, 80, 3010-3014 . RIPLEY, L . S ., 1982, Model for the participation of quasi-palindromic DNA sequences in frameshift mutation . Proceedings of the National Academy of Sciences, USA, 79, 4128-4132 . ROBERTS, J . D ., and KUNKEL, T . A ., 1988, Fidelity of a human cell DNA replication complex . Proceedings of the National Academy of Sciences, USA, 85, 7064-7068 . SANKARANARAYANAN, K ., 1986, Transposable genetic elements, spontaneous mutations and the doubling-dose method of radiation genetic risk evaluation in man . Mutation Research, 160, 73-86 . SARASIN, A ., and BENOIT, A ., 1980, Induction of an error-prone mode of DNA repair in UV-irradiated monkey kidney cells . Mutation Research, 70, 71-81 . SARASIN, A ., and BENOIT, A ., 1986, Enhanced mutagenesis of UV-irradiated simian virus 40 occurs in mitomycin C-treated host cells only at a low multiplicity of infection . Molecular and Cellular Biology, 6, 1102-1107 . SCHAAPER, R . M ., DANFORTH, B . N ., and GLICKMAN, B . W., 1986, Mechanisms of spontaneous mutagenesis : an analysis of the spectrum of spontaneous mutation in the E. coli lac I gene . Journal of Molecular Biology, 189, 273-284 .
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STREISINGER, G ., and OWEN, J . E ., 1985, Mechanism of spontaneous and induced frameshift mutation in bacteriophage T4 . Genetics, 109, 633-659 . SUAREZ, H . G ., DAYA-GROSJEAN, L ., SCHLAIFER, D ., NARDEUX, P ., RENAULT, G ., Bos, J . L., and SARASIN, A., 1989, Activated oncogenes in skin tumors from a repair deficient syndrome, xeroderma pigmentosum . Cancer Research, 49, 1223-1228 . TINDALL, K . R ., and STANKOWSKI JR, L. F ., 1989, Molecular analysis of spontaneous mutations at the gpt locus jp Chinese hamster ovary (AS52) cells . Mutation Research, 220,241-253 . ToozE, J ., 1981, The SV40 nucleotide sequence . DNA Tumour Viruses, edited by J . Tooze (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), pp . 799-804 . YATES, J ., WARREN, N ., REISMAN, D ., and SUGDEN, B ., 1984, Cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells . Proceedings of the National Academy of Sciences, USA . 81, 3806-3810 . ZARBL, H ., SUKUMAR, S ., ARTHUR, A . V ., MARTIN-ZANCA, D ., and BARBACID, M ., 1985, Direct mutagenesis of Ha-ras oncogenes by N-nitroso-N-methyl-urea during initiation of mammary carcinogenesis in rats . Nature (London), 315, 382-385 .
1990,
VOL .
57, NO . 4, 665-676
Mechanisms and consequences of mutation induction in mammalian cells* A . SARASIN, F . BOURRE, L . DAYA-GROSJEAN, A . GENTIL, C . MADZAK and A . STARY
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Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, B .P . n° 8, 94802 Villejuif Cedex, France Mutations have been studied for several decades in order to understand biological processes of great significance and the selection of better-adapted species . Our knowledge both of mutation spectra induced by genotoxic agents and the mechanisms involved in DNA damage processing is more advanced in bacteria than in animal cells . However, the use of new technologies such as shuttle vectors or the polymerase chain reaction will undoubtedly allow rapid progress in the next few years . Shuttle vectors consist of target sequences for monitoring mutagenic activity and additional sequences permitting DNA replication and selection, both in bacteria and in mammalian cells . These plasmids are very efficient in allowing the production of mutation spectra of a particular genotoxin in animal cells . In most cases, base substitutions occur predominantly at the sites of base damage and the type of substitution depends on the kind of damage . This has been well characterized using ultraviolet (UV) light as a mutagen . UV-induced mutations are targeted opposite pyrimidine-pyrimidine sites, where the two major UV lesions are produced . The direct relationships existing between mutation and cancer are exemplified by some hereditary diseases where deficiency in an enzymatic repair system is linked to a high incidence of tumours . Similarly, activation of some cellular proto-oncogenes occurs via specific point mutations . A correlation does exist between the mutation spectra found in model systems and the specific mutation found in the activated oncogene in tumours induced by a given genotoxin . This is particularly well illustrated in the DNA repair deficiency syndrome, xeroderma pigmentosum . The specific mutations found in activated ras oncogenes isolated from UV-stimulated skin tumours correlate well with the mutagenic properties of unrepaired UV-induced DNA lesions . 1.
Introduction Mutations occur in all living systems at various rates depending on the cellular types and the genetic alterations considered . Background mutations are due to unknown causes while induced mutations can be observed after treatment with a genotoxic agent . Most mutations exhibit deleterious effects on the cell or on individuals, but specific genetic changes appear to play a positive role in some basic processes such as species evolution or immunoglobulin gene variability . Numerous types of genetic alterations ranging from large chromosomal changes to a single nucleotide modification have been associated with human diseases such as chromosome gain in Down's syndrome, large deletions in the hereditary retinoblastoma or point mutation in sickle cell anemia . It is clear that a better understanding of the mechanisms of mutation induction will allow a better knowledge of disease appearance leading to more efficient prevention . For obvious *Presented at the 22nd Annual Meeting of the European Society for Radiation Biology, 12-16 September 1989, Brussels . 0020-7616/90 $3 .00 © 1990 Taylor & Francis Ltd
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reasons, point mutations or mutations involving a few bases have been studied in more detail than large genomic rearrangements . Owing to the availability of numerous mutants, the various pathways leading to mutagenesis have been elucidated at the molecular level in E . coli. The absence of well defined mutants in mammalian cells makes it more difficult to study mutagenesis . Moreover, the structure and organization of the mammalian chromatin are so complex that the fate of a given DNA lesion can vary as a function of the cell cycle, the chromosome structure, the transcriptional activity of the gene or the nucleotide sequence . Nevertheless, the various types of DNA lesion processing have been established as shown in figure 1 . In particular, it has been elegantly demonstrated by Hanawalt's group that UV-induced DNA lesions are preferentially repaired in transcriptionally active genes and more on the transcribed strand than on the non-transcribed one (Hanawalt 1987) (figure 1) . Moreover, the mutagenic efficiency of a given lesion will also vary widely as a function of its location in the chromatin structure . Recent advances in technology such as the polymerase chain reaction (PCR) or the use of exogenous probes (animal virus or shuttle vectors) have, however, a
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6 Figure 1 . Various types of DNA lesion processing . DNA lesions are induced on doublestranded DNA due to either `spontaneous' events or genotoxic agents (1) . A complete repair gives rise to the original genetic message (2) while in the absence of any successful repair or tolerance mechanisms, DNA lesions can lead to cellular death (7) . Most DNA lesions represent a block to RNA transcription (3) and DNA replication (5) . Preferential repair has been shown to occur on the transcribed strand (a*) of active genes (4) . DNA lesions on the leading strand constitute a block to replication (5b) while on the lagging strand (5b*) fork displacement may continue . It is believed that trans-lesion synthesis may induce mutation targeted opposite DNA lesions (6) .
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facilitated the determination of mutation spectra in specific genes . PCR is an in vitro method of enzymatic DNA amplification for producing large quantities of a given DNA fragment (Bos et al . 1986) . Hence, PCR followed by DNA sequencing allows a rapid assay for characterizing mutations in DNA isolated from a very small number of mutant cells . This technique has already been used to define spontaneous or induced mutation spectra in integrated DNA sequences (Tindall and Stankowski 1989) . It is obvious that this new technique will be more widely used, and that numerous mutation spectra in a variety of genes will be rapidly obtained . The role of mutations in carcinogenesis has been evoked over several decades, but it is mainly due to the analysis of oncogene activation that a relationship between point mutation and cancer initiation has been demonstrated . Studies on human hereditary diseases such as xeroderma pigmentosum, in which DNA repair deficiency is directly responsible for cancer development, allow a better understanding of the link between DNA lesions, mutation and cancer induction .
2.
Animal virus and transient SV40-shuttle vectors as probes The complex organization of the genome of mammalian cells has necessitated the use of exogenous probes to analyse mutagenesis at the molecular level, The majority of these studies have been carried out with the help of two kinds of probes : animal viruses and shuttle vectors . Among the former, Simian virus 40 (SV40) (Sarasin and Benoit 1980, 1986), Herpes simplex virus (Das Gupta and Summers 1978), adenovirus (Day and Ziolkowski 1981) and parvovirus (Cornelis et al . 1982) have been frequently used as molecular probes to study the effects of physical or chemical DNA-damaging agents . Many experiments have been carried out with SV40 in which permissive simian cells were infected with a temperature-sensitive mutant of SV40 pre-treated in vitro with genotoxic agents . The reversion toward a wild-type phenotype of growth was the mutagenic criterion . The mutants were selected and plaque purified, mutation spectra being established by DNA sequencing (Bourre and Sarasin 1983, Gentil et al. 1986) . More recently, vectors able to replicate in mammalian cells as well as in bacteria were developed . These shuttle vectors are grown in mammalian cells and subsequently screened for mutation in bacteria . Numerous shuttle vectors have been constructed and most of them are SV40-based . Typically, as shown in figure 2, a shuttle vector carries two origins of replication : a mammalian one derived from a eukaryotic virus (EBV, SV40), allowing replication of the vector in mammalian cells, and a plasmidic one, allowing replication in bacteria . Plasmid DNA is treated in vitro with DNA-damaging agents prior to transfection in mammalian cells . After its replication the plasmid DNA is harvested from the cells where the mutations occur and screened in bacteria . Mutants are selected in the target gene (sup F, lac I etc) and the mutation spectra are determined by DNA sequencing (Calos et al. 1983, Razzaque et al. 1983) . The two biological models present some advantages and disadvantages . Indeed, transient shuttle vectors allow analysis at the molecular level of a great number of mutants selected in a forward assay, whereas with the animal viruses only a small number of mutants are analysed by a time-consuming reversion assay . One of the main disadvantages in the use of shuttle vectors is the requirement of DNA transfection to introduce the mutational target into the cells, while the animal viruses are introduced into host cells by natural infection . When using DNA transfection only 5-10 per cent of the cells effectively take up the DNA, and it is not
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Figure 2 . Schematic representation of a shuttle vector genetic map . Mammalian ORI (SV40, Py or EBV derived) is a DNA sequence from animal virus replication origin allowing replication in mammalian cells by using a viral Trans acting protein (SV40 TAg, Py TAg or EBNA1 protein) . Bacterial ORI is a DNA sequence from the replication origin of the pBR plasmid . Selection gene (resistance to G418 or hygromycin) and Antibiotic resistance gene (Amp, Tet, Cm or Neo) code for proteins allowing selection of the plasmids respectively in mammalian cells and in bacteria . Target gene (gal K, lac I, lac Z, sup F, lac 0, HSV-tk, gpt or aprt) represents sequence used for screening mutations .
known whether these cells represent either a random part of the cell population or a subpopulation in a different physiological state . Moreover, this step may be responsible for the higher background mutation frequency observed with transient shuttle vectors compared with that measured with animal viruses or endogenous genes (for review see Bourre et al . 1989b) . To avoid transfection problems Yates et al. (1984) reported on the use of Epstein-Barr virus episomal shuttle vectors . These plasmids are maintained stably in human cells and can be rescued in bacteria to screen mutagenesis . Nevertheless, they are technically difficult to work with, and it is not possible to treat them independently of the cells . 3 . Origins of mutation 3 .1 Spontaneous mutation Spanning the range from bacteria to higher eukaryotes, the mutation rate is remarkably low, between 10 -9 and 10 -12 errors per base pair per generation (Cox 1976) . This low spontaneous mutation frequency could result from various processes . In vitro studies show that the accuracy of eukaryotic DNA polymerases depends on the kind of polymerase, the type of 'mispair' that is formed and the surrounding
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sequence (Lai and Beattie 1988) . Error rates measured in in vitro replication systems remain much higher than for chromosomal loci in vivo (Roberts and Kunkel 1988), suggesting that additional fidelity factors, such as the proofreading activity, may be active during chromosomal replication . Moreover, mismatch repair appears to be very efficient in vivo on newly synthesized DNA . Despite these cellular mechanisms, spontaneous mutations appear during DNA replication . They could also be due to spontaneous chemical modifications of the DNA . In particular it has been shown that 10,000 depurinations per day occur in a mammalian cell (Lindahl 1982) . They are due to the spontaneous hydrolysis of the N-glycosilic bond or appear as intermediates in DNA repair . Apurinic sites (Ap) are recognized and repaired by specific Ap-endonucleases, ubiquitous in both prokaryotes and eukaryotes (Friedberg 1985) . This might indicate the high toxicity of unrepaired Ap sites . Indeed, it has been shown in mammalian cells that the presence of Ap sites strongly decreases the survival of treated SV40 virus, -and that they are highly mutagenic (Gentil et al . 1984) . Another possible cause for background mutations is the spontaneous de-amination of cytosine, especially of 5-methyl cytosine into thymine (Bird 1980) . 5-mC is found in the dinucleotide CpG which is the major methylated sequence in animals . The implication of mCpG in spontaneous mutagenesis is supported by studies comparing the sequences of 5 S RNAs of Xenopus laevis (Brownlee et al . 1972) or of the chicken ovalbumin gene (Mandel and Chambon 1979) . Among the factors favouring the occurrence of mutations it has been proposed that secondary structures could play a role . Indeed, in E . coli, frameshifts occur frequently at reiterated base sequences (Streisinger and Owen 1985) and addition/deletion events concern mostly repeated units (Schaaper et al . 1986) suggesting a misaligned pre-mutational intermediate containing an (some) extrahelical nucleotide(s) . Similarly, deletions account for 10 per cent of the spontaneous mutants at the aprt locus of hamster cells, and occur by non-homologous recombination between short sequence repeats (Nalbantoglu et al . 1986) . Ripley has proposed that the in vivo processing of misaligned quasipalindromic DNA sequences could produce frameshift and base substitution mutations . Analyses of spontaneous frameshifts in E . coli (De Boer and Ripley 1984) and in Saccharomyces cerevisiae (Ripley 1982) correlate with this hypothesis . Finally, it has been proposed that transposable genetic elements would be responsible for numerous mutations, although the mechanisms by which they occur are not known (for review see Sankaranarayanan, 1986) .
3 .2
Induced mutation It is now well known that DNA-damaging agents induce mutations . Therefore it is important to define the location of the induced mutation in relation to the lesion produced, which permits one to estimate which lesions are pre-mutagenic . Among DNA damaging agents, 254nm ultraviolet (UV) light in particular has been extensively analysed, since it represents one of the major carcinogens in humans . UV-induced mutations in mammalian cells were found to occur almost exclusively at dipyrimidine sequences in studies with SV40 virus (Bourre and Sarasin 1983, Braggaar et al . 1985), shuttle vectors (Hauser et al . 1986, Hsia et al. 1989) or at endogenous loci (Drobetsky et al . 1987) . These sequences being potential UVinduced lesion sites, mutagenesis appears to be essentially targeted (figure 3) . The main lesions induced by UV-irradiation on DNA are the major cy-
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1000-fold elevated skin cancer incidence is due to the complete or partial absence of repair in UV-damaged DNA, particularly of pyrimidine dimers and pyrimidine (6-4) pyrimidones (figure 1) . We have analysed DNA from 12 epithelial tumours (basal or squamous cell carcinomas) obtained from young XP patients . DNA from two independent tumours from the same XP child were able to form foci in the 3T3 cells due to the transforming activity of the N-ras gene (Suarez et al. 1989) . Using the polymerase chain reaction for gene amplification and differential hybridization with synthetic oligonucleotide probes (Bos et al . 1986) we were able to determine the presence of a critical point mutation in codon 61 of the N-ras gene (Suarez et al . 1989) . This results in the substitution of a glutamine by a histidine residue which is sufficient to activate the Ras protein (figure 4) . Recently a tumour from another XP patient was found to contain an activated Ki-ras gene, due to a point mutation in codon 12 resulting in the substitution of a glycine by a valine (figure 4) . We interpret these results in the model depicted in figure 4 : UV-irradiation of the epithelial cells in the exposed areas of the skin induces lesions which if unrepaired could lead after replication to the incorporation of a wrong base opposite the pyrimidine involved in the lesion . Hence, the XP syndrome represents a human cancer model comparable to the animal tumour studies where oncogene activation by a point mutation is correlated with a specific type of adduct present on the cellular DNA . However, as most neoplasms develop with a long latent period and go through a series of progressive pathological stages, oncogenesis must involve more than a simple activation of a single transforming gene . For example, benign skin papilloma induced in animals or in humans contain activated ras genes (often Ha-ras) but the majority of these tumours regress and never give rise to cancers (Balmain et al. 1984, Corominas et al . 1989) . Secondary genetic events therefore seem necessary for tumour progression . Hence, we screened the XP tumours for the existence of other genetic modifications, particularly of other oncogenes . Indeed, the majority of the tumours exhibit amplification of the myc and/or Ha-ras oncogene . In particular, the Ha-ras gene was amplified and often rearranged in over 40 per cent of the tumours, a level significantly higher than seen (1 per cent) in human tumours in general . This high level of amplification in XP cells is probably due to UV-induced lesions blocking replication and resulting in multiple cycles of abortive reinitiation (Lavi 1981) . This type of random amplification in genes such as myc or Ha-ras
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Figure 4 . A model for proto-oncogene activation by point mutation in UV-induced epithelioma from xeroderma pigmentosum . Irradiation by UV light induces the formation of pyrimidine dimers or of pyrimidine(6-4)pyrimidone photoproducts (A) . In XP cells these lesions are not repaired and replication of the damaged DNA results in a point mutation opposite the lesion . When this occurs in a crucial codon of a protooncogene, such as codon 61 of the N-ras gene or codon 12 of the Ki-ras gene, the modification of the genetic code results in the activation of the gene (transforming activity) . Numbers correspond to the nucleotides in the coding sequence .
probably leads to selective cell proliferation which, together with activation of a second oncogene (e .g . N-ras), permits progression to a fully transformed phenotype . The results obtained from our study of XP tumours have substantiated the hypothesis made in animal studies as applicable to human carcinogenesis . A direct correlation is now possible between the mutation spectrum induced by a specific genetic lesion and the types of mutations observed in activated oncogenes . Acknowledgements This work was supported by grants from the Commission of the European Communities, Brussels, Belgium (n . B16-E-163 F), from the Ministere de la Recherche et de l'Enseignement Superieur and from the Association pour la Recherche sur le Cancer, Villejuif, France .
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References BALMAIN, A ., and BROWN, K ., 1988, Oncogene activation in chemical carcinogenesis . Advances in Cancer Research, 51, 147-182 . BALMAIN, A., RAMSDEN, M ., BOWDEN, G . T., and SMITH, J ., 1984, Activation of the mouse cellular Harvey-ras gene in chemically induced benign skin papilloma . Nature (London), 307, 658-660. BARBACID, M ., 1987, Ras genes . Annual Review of Biochemistry, 56, 779-827 . BIRD, A . P ., 1980, DNA methylation and the frequency of CpG in animal DNA . Nucleic Acids Research, 8, 1499-1503 . Bos, J . L ., VERLAAN-DE-VRIES, M ., MARSHALL, C . J ., VEENEMAN, G . H ., VAN Boom, J . H ., and VAN DER EB, A ., 1986, Human gastric carcinoma contains a single mutated and an amplified normal allele of the Ki-ras oncogene . Nucleic Acids Research, 14, 1209-1217 . BOURRE, F., and SARASIN, A ., 1983, Targeted mutagenesis of SV40 DNA induced by UV light . Nature (London), 305, 68-70 . BOURRE, F., BENOIT, A ., and SARASIN, A., 1989a, Respective role of pyrimidine dimer and pyrimidine(6-4)pyrimidone photoproducts in UV mutagenesis of SV40 DNA in mammalian cells . Journal of Virology, 63, 4520-4524 . BOURRE, F ., RENAULT, G ., AND GENTIL, A ., 1989b, From Simian Virus 40 to transient shuttle vectors in mutagenesis studies . Mutation Research, 220, 107-113 . BRAGGAAR, H ., CORNELIS, J . J ., VAN DER LUBBE, J . L . M ., and VAN DER EB, A . J ., 1985, Mutagenesis in UV-irradiated Simian virus 40 occurs predominantly at pyrimidine doublets . Mutation Research, 142, 75-81 . BRASH, D . E ., SEETHARAM, S ., KRAEMER, K . H ., SEIDMAN, M . M ., and BREDBERG, A ., 1987, Photoproduct frequency is not the major determinant of UV base substitution hot spots or cold spots in human cells . Proceedings of the National Academy of Sciences, USA, 84, 3782-3786 . BROWNLEE, G . G ., CARTWRIGHT, E ., MCSHANE, T ., and WILLIAMSON, R ., 1972, The nucleotide sequence of somatic 5S RNA from Xenopus lcevis . FEBS Letters, 25, 8-12 . BURNOUF, D ., KOEHL, P ., and FUCHS, R. P . P ., 1989, Single adduct mutagenesis : strong effect of the position of a single acetylaminofluorene adduct within a mutation hot spot . Proceedings of the National Academy of Sciences, USA, 86, 4147-4151 . CALOS, M . P ., LEBKOWSKI, J . S ., and BOTCHAN, M . R ., 1983, High mutation frequency in DNA transfected into mammalian cells . Proceedings of the National Academy of Sciences, USA, 80, 3015-3019 . CLEAVER, J . E ., 1968 . Defective repair replication of DNA in xeroderma pigmentosum . Nature (London), 218, 652-656 . CORNELIS, J . J ., Su, Z . Z ., and ROMMELAERE, J ., 1982, Direct and indirect effects of UV-light on the mutagenesis of parvovirus H-1 . EMBO Journal, 1, 693-699 . COROMINAS, M ., KAMINO, H ., LEON, J ., and PELLICER, A ., 1989, Oncogene activation in human benign tumors of the skin (keratoacanthomas) : is ras involved in differentiation as well as proliferation? Proceedings of the National Academy of Sciences, USA, 86,6372-6376 . Cox, E . C ., 1976, Bacterial mutator genes and the control of spontaneous mutation . Annual Review of Genetics, 10, 135-156 . DAs GUPTA, U . B ., and SUMMERS, W . C ., 1978, Ultraviolet reactivation of Herpes simplex virus is mutagenic and inducible in mamalian cells . Proceedings of the National Academy of Sciences, USA, 75, 2378-2381 . DAY III, R . S ., and ZIOLKOWSKI, C . H . J ., 1981, UV-induced reversion of adenovirus 5 ts2 infecting human cells . Photochemistry and Photobiology, 34, 403-406 . DE BOER, J . G ., and RIPLEY, L . S ., 1984, Demonstration of the production of frameshift and base-substitution mutations by quasipalindrotnic DNA sequences . Proceedings of the National Academy of Sciences, USA, 81, 5528-5531 . DROBETSKY, E . A ., GROSOVSKY, A . J ., and GLICKMAN, B . W ., 1987, The specificity of UV induced mutations at an endogenous locus in mammalian cells . Proceedings of the National Academy of Sciences, USA, 84, 9103-9107 . FRIEDBERG, E . C ., 1985, DNA Repair (New York : Freeman) .
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FUCHS, R . P . P ., 1983, DNA binding spectrum of the carcinogen N-acetoxy-N-2acetylaminofluorene significantly differs from the mutation spectrum . Journal of Molecular Biology, 177,173-180 . FUCHS, R . P . P ., LEFEVRE, J . F ., POUYET, J ., and DAUNE, M . P ., 1976, Comparative orientation of the fluorene residue in native DNA modified by N-acetoxy-N-2acetylaminofluorene and two 7-halogeno derivatives . Biochemistry, 15, 3347-3351 . GENTIL, A ., MARGOT, A ., and SARASIN, A ., 1984, Apurinic sites cause mutations in simian virus 40 . Mutation Research, 129, 141-147 . GENTIL, A ., MARGOT, A ., and SARASIN, A ., 1986, 2-(N-acetoxy-N-acetylamino)fluorene mutagenesis in mammalian cells : sequence-specific hot spot. Proceedings of the National Academy of Sciences, USA, 83, 9556-9560 . HANAWALT, P . C ., 1987, Preferential DNA repair in expressed genes . Environmental Health Perspectives, 76, 9-14 . HANAWALT, P . C ., and SARASIN, A ., 1986, Cancer prone hereditary diseases with DNA processing abnormalities . Trends in Genetics, 2, 124-129 . HAUSER, J ., SEIDMAN, M . M ., SIDUR, K ., and DIXON, K ., 1986, Sequence specificity of point mutations induced during passage of a UV-irradiated shuttle vector plasmid in monkey cells . Molecular and Cellular Biology, 6, 277-285 . HSIA, H . C ., LEBKOWSKI, J . S ., LEONG, P . M ., CALOS, M . P., and MILLER, J . H ., 1989, Comparison of ultraviolet irradiation-induced mutagenesis of the lacl gene in Escherichia coli and in human 293 cells . Journal of Molecular Biology, 205, 103-113 . KNUDSON, A . G ., 1986, Genetics of human cancer . Annual Review of Genetics, 20, 231-251 . KOEHL, P ., BURNOUF, D ., and FUCHS, R . P . P ., 1989, Construction of plasmids containing a unique acetylaminofluorene adduct located within a mutation hot spot . A new probe for frameshift mutagenesis . Journal of Molecular Biology, 207, 355-364 . LAI, M .-D ., and BEATTIE, K . L ., 1988, Influence of DNA sequence on the nature of mispairing during DNA synthesis . Biochemistry, 27, 1722-1728 . LAVI, S ., 1981, Carcinogen-mediated amplification of viral DNA sequences in simian virus 40-transformed Chinese hamster embryo cells . Proceedings of the National Academy of Sciences, USA, 78, 6144-6148 . LINDAHL, T ., 1982, DNA repair enzymes . Annual Review of Biochemistry, 51, 61-87 . MANDEL, J . L., and CHAMBON, P ., 1979, DNA methylation : organ specific variations in the methylation pattern within and around ovalbumin and other chicken genes . Nucleic Acids Research, 7, 2081-2103 . NALBANTOGLU, J ., HARTLEY, D ., PHEAR, G ., TEAR, G ., and MEUTH, M ., 1986, Spontaneous deletion formation at the aprt locus of hamster cells : the presence of short sequence homologies and dyad symmetries at deletion termini . EMBO Journal, 5, 1199-1204 . PROTIC-SABLJIC, M ., TUTEJA, N ., MUNSON, P . J ., HAUSER, J ., KRAEMER, K . H ., and DIXON, K ., 1986, UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells. Molecular and Cellular Biology, 6, 3349-3356 . RAZZAQUE, A ., MIZUSAWA, H ., and SEIDMAN, M . M ., 1983, Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells . Proceedings of the National Academy of Sciences, USA, 80, 3010-3014 . RIPLEY, L . S ., 1982, Model for the participation of quasi-palindromic DNA sequences in frameshift mutation . Proceedings of the National Academy of Sciences, USA, 79, 4128-4132 . ROBERTS, J . D ., and KUNKEL, T . A ., 1988, Fidelity of a human cell DNA replication complex . Proceedings of the National Academy of Sciences, USA, 85, 7064-7068 . SANKARANARAYANAN, K ., 1986, Transposable genetic elements, spontaneous mutations and the doubling-dose method of radiation genetic risk evaluation in man . Mutation Research, 160, 73-86 . SARASIN, A ., and BENOIT, A ., 1980, Induction of an error-prone mode of DNA repair in UV-irradiated monkey kidney cells . Mutation Research, 70, 71-81 . SARASIN, A ., and BENOIT, A ., 1986, Enhanced mutagenesis of UV-irradiated simian virus 40 occurs in mitomycin C-treated host cells only at a low multiplicity of infection . Molecular and Cellular Biology, 6, 1102-1107 . SCHAAPER, R . M ., DANFORTH, B . N ., and GLICKMAN, B . W., 1986, Mechanisms of spontaneous mutagenesis : an analysis of the spectrum of spontaneous mutation in the E. coli lac I gene . Journal of Molecular Biology, 189, 273-284 .
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STREISINGER, G ., and OWEN, J . E ., 1985, Mechanism of spontaneous and induced frameshift mutation in bacteriophage T4 . Genetics, 109, 633-659 . SUAREZ, H . G ., DAYA-GROSJEAN, L ., SCHLAIFER, D ., NARDEUX, P ., RENAULT, G ., Bos, J . L., and SARASIN, A., 1989, Activated oncogenes in skin tumors from a repair deficient syndrome, xeroderma pigmentosum . Cancer Research, 49, 1223-1228 . TINDALL, K . R ., and STANKOWSKI JR, L. F ., 1989, Molecular analysis of spontaneous mutations at the gpt locus jp Chinese hamster ovary (AS52) cells . Mutation Research, 220,241-253 . ToozE, J ., 1981, The SV40 nucleotide sequence . DNA Tumour Viruses, edited by J . Tooze (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York), pp . 799-804 . YATES, J ., WARREN, N ., REISMAN, D ., and SUGDEN, B ., 1984, Cis-acting element from the Epstein-Barr viral genome that permits stable replication of recombinant plasmids in latently infected cells . Proceedings of the National Academy of Sciences, USA . 81, 3806-3810 . ZARBL, H ., SUKUMAR, S ., ARTHUR, A . V ., MARTIN-ZANCA, D ., and BARBACID, M ., 1985, Direct mutagenesis of Ha-ras oncogenes by N-nitroso-N-methyl-urea during initiation of mammary carcinogenesis in rats . Nature (London), 315, 382-385 .
