Mutagenic properties of the 8-amino-2'-deoxyguanosine DNA adduct in mammalian cells.

The DNA adduct 8-amino-2'-deoxyguanosine (8-amino-dG) is found in liver DNA of rats treated with the hepatocarcinogen 2-nitropropane. Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of 8-amino-dG in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'-TCCTCCTX1G2CCTCTC and 5'-TCCTCCTG1X2CCTCTC, X = dG or 8-amino-dG) with the lesion positioned at codon 60 or 61 of the non-coding strand of the human c-Ha- ras1 gene were inserted into single-stranded phagemid vectors and transfected into COS-7 cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequencing to establish the mutation frequency and spectrum produced by the modified base. The correct base, dCMP, was incorporated preferentially opposite 8-amino-dG at X1and X2. When 8-amino-dG was at X1, targeted GNH2-->T transversions were detected, along with smaller numbers of GNH2-->A transitions and GNH2-->C transversions. When the adduct was at X2, only GNH2-->T transversions were observed. The frequencies of targeted mutation at X1and X2were 2.7 and 1.7%, respectively. Mutation frequency and mutagenic spectrum were sequence context dependent. In addition, non-targeted G-->T transversions, accompanied by some G-->A transitions, were detected 5' to 8-amino-dG when the lesion was at X2. We conclude that 8-amino-dG is a mutagenic lesion, generating G-->T and G-->C transversions and G-->A transitions in mammalian cells.     

Mutagenic properties of 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene, a persistent acetylaminofluorene-derived DNA adduct in mammalian cells

The carcinogen 2-acetylaminofluorene is metabolically activated in cells and reacts with DNA to form N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF), N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF), and 3-(deoxyguanosin-N(2)()-yl)-2-acetylaminofluorene (dG-N(2)-AAF) DNA adducts. The dG-N(2)-AAF adduct is the least abundant of the three isomers, but it persists in the tissues of animals treated with this carcinogen. The miscoding and mutagenic properties of dG-C8-AAF and dG-C8-AF have been established; these adducts are readily excised by DNA repair enzymes engaged in nucleotide excision repair. In the present study, oligodeoxynucleotides modified site-specifically with dG-N(2)-AAF were used as DNA templates in primer extension reactions catalyzed by mammalian DNA polymerases. Reactions catalyzed by pol alpha were strongly blocked at a position one base before dG-N(2)-AAF and also opposite this lesion. In contrast, during translesion synthesis catalyzed by pol eta or pol kappa nucleotides were incorporated opposite the lesion. Both pol eta and pol kappa incorporated dCMP, the correct base, opposite dG-N(2)-AAF. In reactions catalyzed by pol eta, small amounts of dAMP misincorporation and one-base deletions were detected at the lesion site. With pol kappa, significant dTMP misincorporation was observed opposite the lesion. Steady-state kinetic analysis confirmed the results obtained from primer extension studies. Single-stranded shuttle vectors containing (5)(')TCCTCCTCXCCTCTC (X = dG-N(2)-AAF, dG-C8-AAF, or dG) were used to establish the frequency and specificity of dG-N(2)-AAF-induced mutations in simian kidney (COS-7) cells. Both lesions promote G --> T transversions overall, with dG-N(2)-AAF being less mutagenic than dG-C8-AAF (3.4% vs 12.5%). We conclude from this study that dG-N(2)-AAF, by virtue of its persistence in tissues, contributes significantly to the mutational spectra observed in AAF-induced mutagenesis and that pol eta, but not pol kappa, may play a role in this process.     

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

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

Two distinct translesion synthesis pathways across a lipid peroxidation-derived DNA adduct in mammalian cells

Translesion DNA synthesis (TLS) of damaged DNA templates is catalyzed by specialized DNA polymerases. To probe the cellular TLS mechanism, a host-vector system consisting of mouse fibroblasts and a replicating plasmid bearing a single DNA adduct was developed. This system was used to explore the TLS mechanism of a heptanone-etheno-dC (H-epsilondC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-epsilondC almost exclusively directed incorporation of dT and dA. Whereas knockout of the Y family TLS polymerase genes, Polh, Polk, or Poli, did not qualitatively affect these TLS events, inactivation of the Rev3 gene coding for a subunit of polymerase zeta or of the Rev1 gene abolished TLS associated with dA, but not dT, insertion. The analysis of results of the cellular studies and in vitro TLS studies using purified polymerases has revealed that the insertion of dA and dT was catalyzed by different polymerases in cells. While insertion of dT can be catalyzed by polymerase eta, kappa, and iota, insertion of dA is catalyzed by an unidentified polymerase that cannot catalyze extension from the resulting dA terminus. Therefore, the extension from this terminus requires the activity of polymerase zeta-REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block.     

Mutagenic effects of 8-hydroxy-dGTP in live mammalian cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP), was examined using COS-7 cells. 8-OH-dGTP and supF shuttle plasmid DNA were cointroduced by means of cationic liposomes, and the DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T-->C:G substitution mutations in the COS-7 cells. This result agrees with previous observations indicating that DNA polymerases misincorporate 8-OH-dGTP opposite A in vitro, and that the oxidized deoxyribonucleotide induces A:T-->C:G transversions in E. coli. These results constitute the first direct evidence to show that 8-OH-dGTP actually induces mutations in living mammalian cells.     

Mutagenic properties of a unique abasic site in mammalian cells

The mutagenic properties of a true unique abasic site located opposite a guanine residue were studied. An oligonucleotide containing a chemically-produced abasic site was inserted into a shuttle vector able to replicate both in simian cells and in bacteria. Plasmid DNA was rescued from simian cells and screened in bacteria by differential hybridization with a labelled oligonucleotide probe. Mutations were easily detected and sequenced. Results showed that opposite a guanine the abasic site was error free repaired or replicated by mammalian cells with an efficiency of 99%. Point mutations occurred at a frequency of approximately 1% in control host cells and at more than 3% in UV-pre-irradiated host cells. Adenine, cytosine or thymine were found to have been inserted opposite the abasic site. No preferential insertion for a particular base was observed in contrast to that reported in bacteria.     

Translesion synthesis past 2'-deoxyxanthosine, a nitric oxide-derived DNA adduct, by mammalian DNA polymerases

Cellular DNA is damaged by nitric oxide (NO), a multifunctional bioregulator and an environmental pollutant that has been implicated in diseases associated with cancer and chronic inflammation. 2'-Deoxyxanthosine (dX) is a major NO-derived DNA lesion. To explore the mutagenic potential of dX, a 38-mer oligodeoxynucleotide ((5')CATGCTGATGAATTCCTTCXCTTCTTTCCTCTCCCTTT) modified site-specifically with dX at the X position was prepared post-synthetically and used as a DNA template in primer extension reactions catalyzed by calf thymus DNA polymerase (pol) alpha and human DNA pol beta, eta, and kappa. Primer extension reactions catalyzed by pol alpha or beta in the presence of four dNTPs were retarded at the dX lesion while pol eta and kappa readily bypassed the lesion. The fully extended products were analyzed to quantify the miscoding specificity and frequency of dX using two-phase polyacrylamide gel electrophoresis (PAGE). With pol alpha, eta and kappa, incorrect dTMP was preferentially incorporated opposite the lesion, along with lesser amounts of dCMP, the correct base. When pol beta was used, direct incorporation of correct dCMP was primarily observed, accompanied by small amounts of misincorporation of dTMP, dAMP and dGMP. Steady-state kinetic analyses supported the results obtained from the two-phase PAGE assay. dX is a miscoding lesion capable of preferentially generating G-->A mutations. The miscoding frequency varied depending on DNA polymerase used.     

Mutagenic specificity in reovirus.

Mutagenic specificity in response to chemical mutagens has been observed with certain temperature-sensitive mutants of reovirus type 3. One mutant induced by nitrous acid reverted specifically with nitrous acid. Three nitrosoguanidine-induced mutants reverted after nitrous acid treatment. These findings thus suggest that analysis of chemical induction of reversion from the temperature-sensitive phenotype may be a useful approach for studying the nature of mutation in animal viruses.     

Supercoiling-dependent sequence specificity of mammalian DNA methyltransferase.

Negative supercoiling of substrate DNA dramatically alters the in vitro sequence specificity of mammalian DNA methyltransferase (DNA MeTase). This result suggests that in vivo site selection by DNA MeTase could be regulated by conformational information in the form of alternative secondary structures induced in DNA by local supercoiling or by the binding of specific nuclear proteins. DNA in the left-handed Z-form is shown not to be a substrate for mammalian DNA MeTase. The sensitivity of DNA MeTase to DNA structure may also make it useful as a probe for sequences which undergo supercoiling-dependent structural transitions in vitro.     

Mutagenic specificity of ultraviolet light

Genetic and sequencing studies of ultraviolet light (u.v.)-induced mutations in the lacI gene of Escherichia coli show the following: u.v. stimulates many types of mutations. In lacI, base substitutions account for 60 to 65% of the observed mutations, small frameshifts 30 to 35%, and deletions of more than several base-pairs approximately 5%. A comparison of the mutational spectrum of u.v.-induced mutations with those of other SOS-dependent mutagens and with the mutations produced by inducing the SOS system in the absence of mutagenic treatment indicates that most u.v.-induced base substitutions are "targeted", resulting from premutational lesions across from the site of the mutations. Among base substitutions, both transitions and transversions occur, although the most favored mutational sites involve G X C----A X T transitions. G X C----A X T transitions are induced preferentially at sites of adjacent pyrimidines. In one case the conversion of a site from -A-C-A- to -T-C-A- results in a 15-fold increase in u.v.-induced C----T transitions. Frameshifts at certain sites are well-induced by u.v., and the largest hotspot in the I gene involves the loss of an (sequence in text) base pair from a (sequence in text) sequence. Of 25 frameshifts detected by DNA sequencing, 23 mutations at seven different sites result from the elimination of a single base-pair, and two mutations result from the elimination of two base-pairs. No additions were detected. The use of a lacI-Z fusion system, which allows direct selection of frameshifts of either sign, reveals that throughout the entire gene frameshifts that eliminate a single base-pair (-1) predominate by a factor of 20 or more over frameshifts that add a single base-pair (+1). In one case a two-base-pair elimination occurs frequently, resulting in the loss of a -C-T- sequence (on one strand), or a -T-C- sequence, from a -C-T-C-T-C-T-C- sequence. For both frameshifts and base substitutions, some aspect of the larger surrounding sequence beyond the nearest neighbors can influence mutation rates by as much as 50-fold, thus determining which sites are seen as hotspots. The bearing of these and other data on the detailed mechanism of mutagenesis is considered in the Discussion.     

Quantification of aminofluorene adduct formation and repair in defined DNA sequences in mammalian cells using the UVRABC nuclease

Using the UVRABC nuclease as a reagent coupled with DNA restriction and hybridization analysis we have developed a method to quantify N-acetoxy-2-acetylaminofluorene (NAAAF)-induced DNA damage in the coding and noncoding sequences of the dihydrofolate reductase (DHFR) gene in Chinese hamster ovary (CHO) cells. High performance liquid chromatography analysis shows that the only DNA adduct formed in NAAAF-treated CHO cells is N-(deoxyguanosine-C8-yl)-2-aminofluorene (dG-C8-AF). DNA sequencing analysis demonstrates that the UVRABC nuclease incises at all potential sites in which dG-C8-AF adduct may form in linear DNA fragments. We have found that the formation and removal of dG-C8-AF adducts in the coding and 3' downstream noncoding sequences of the DHFR domain are similar in cells treated with 10 microM NAAAF (3.1 adducts/14 kilobases); DNA adduct removal attains 70% for both sequences within 24 h. This result contrasts with that obtained for the repair of cyclobutane dipyrimidines in the DHFR gene, in which the repair efficiency is much higher in the coding region than in the 3' downstream noncoding region. Our results suggest that in CHO cells the repair pathway for aminofluorene DNA adducts is not the same as that for cyclobutane dipyrimidines. This new technique has the potential to detect a variety of chemical carcinogen induced DNA adducts at the gene level in cultured cells and in DNA isolated from animal tissues.     

Mutagenic specificity of imidazole ring-opened 7-methylpurines in M13mp18 phage DNA

The most abundant lesion formed in DNA upon modification with methylating agents 7-methylguanine, under alkaline conditions is converted into 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeGua). We have previously shown that treatment of dimethylsulfate methylated DNA with NaOH creates mutagenic base derivatives leading to a 60-fold increase in the frequency of A-->G transitions and a 2-3-fold increase of G-->T and G-->C transversions. We have analyzed which lesions lead to these mutations. We compared mutagenic spectra in the lacZ gene of M13mp18 phage DNA modified with dimethylsulfate and NaOH after selective elimination of damaged bases from molecules used for transfection into SOS-induced E. coli. Partial elimination of Fapy-7MeGua from phage DNA performed by its digestion with formamidopyrimidine-DNA glycosylase resulted in a 2-3-fold decrease of G-->T and G-->C transversions. Selective depurination of methylated bases (9 h, 37 degrees C, pH 7.0) resulting in almost complete loss of 7MeAde as demonstrated by HPLC analysis of [3H]MNU alkylated phage DNA used as a probe, caused a dramatic, 9-fold decrease of A-->G transitions. Alkali-catalysed rearrangement of 7MeAde was followed by HPLC analysis of [3H]MNU alkylated poly(A) and poly(dA). After incubation of these oligonucleotides in NaOH, 7MeAde disappeared from both chromatograms, but only in polyA, 2 new peaks migrating with retention time different from that of 1MeAde, 3MeAde or 7MeAde were detected, suggesting formation of two rotameric forms of Fapy-7MeAde as observed for Fapy-7MeGua. Thus the miscoding lesion, giving rise to A-->G transitions derived from 7MeAde was Fapy-7MeAde. Fapy-7MeGua was at least an order of magnitude less mutagenic, but in SOS-induced cells it gave rise to G-->T and G-->C transversions.     

The sequence specificity of a mammalian DNA methylase.

The sequence specificity of an extensively purified DNA methylase preparation from Krebs II mouse ascites cells has been examined. The enzyme appears to be highly sequence dependent. Moreover the sequence distribution of cytosine residues that are methylated, bears a very close resemblance to the sequence distribution of 5'-methyl cytosine found in vivo in a wide range of vertebrate cells and is consistent with methylation of cytosines in the sequence R-Yn-C-R.     

Relationship between mutagenicity and DNA adduct formation in mammalian cells for fjord- and bay-region diol-epoxides of polycyclic aromatic hydrocarbons

Chinese hamster V79 cells were treated with the anti- and syn-diastereomers of the bay- or fjord-region diol-epoxides of four polycyclic aromatic hydrocarbons, namely benzo[a]pyrene (BP), benzo[c]chrysene (BcC), benzo[g]chrysene (BgC) and benzo[c]phenanthrene (BcPh). The frequency of induction of 6-thioguanine-resistant mutations was determined, and the extent of formation of DNA adducts was measured by 32P-postlabelling. When expressed as mutation frequency per nanomoles compound per millilitre incubation medium, this group of chemicals expressed a 160-fold range in potency. In agreement with previous experimental studies, the anti-diol-epoxide of BcC was highly mutagenic, inducing in excess of 3 x 10(4) mutations/10(6) cells per nmol compound/ml. The mutagenic activities of the anti- and syn-diol-epoxides of BP were 10- and 100-fold lower, respectively. Both diol-epoxides of BgC, the syn-BcC and the anti-BcPh derivatives were also highly mutagenic, and only the syn-BcPh diol-epoxide was less mutagenic than the anti-diol-epoxide of BP. Determination of the levels of DNA adducts formed by the diol-epoxides indicated that the most mutagenic compounds were the most DNA reactive, although the fjord-region diol-epoxides gave rise to more complex patterns of adducts than those of the BP diol-epoxides. When the mutagenicity results were expressed as mutations per femtomoles total adducts formed, all compounds showed similar activities. Thus the potent mutagenicity of the fjord region diol-epoxides appears to be due to the high frequency with which they form DNA adducts in V79 cells, rather than to formation of adducts with greater mutagenic potential.