Mutation spectrum of copper-induced DNA damage.

The ability of metal ions to damage DNA and cause mutagenesis has been analyzed with reversion and forward mutation assays using single-stranded DNA templates. We previously reported that incubation of phi X174 am3 DNA with Fe2+ in vitro results in mutagenesis when the treated DNA is transfected into Escherichia coli spheroplasts (Loeb, L. A., James, E. A., Waltersdorph, A. M., and Klebanoff, S. J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3918-3922, 1988). We now extend these studies to other metal ions. Of the metal ions tested, copper ions were the most mutagenic; the frequency of mutants produced was equal to or greater than that produced by Fe2+. Mutagenesis by Cu+ was diminished by catalase, mannitol, and superoxide dismutase suggesting the involvement of H2O2, hydroxyl ions, and superoxide, respectively. However, the findings that Cu+ and Cu2+ are nearly equally mutagenic and that the mutagenic activities are not completely inhibited by oxygen free radical scavengers make it unlikely that the mechanism for mutagenesis is simply the production of hydroxyl free radicals. The spectra of mutations produced by either copper ion using the lacZ gene as a target are very similar and differ from those reported with other agents. The predominant mutagenic sequence changes are single-base substitutions, the most frequent being replacement of a template C by a T. This transition presumably results from mispairing of an altered C with deoxyadenosine. Copper-induced mutations are not randomly distributed. Instead, they are found predominantly in clusters suggesting direct interaction of copper ions with specific nucleotide sequences in DNA. Evidence is considered that the high frequency of C----T transitions may be a common manifestation of DNA damage by oxygen radicals.     

Glyoxal, a major product of DNA oxidation, induces mutations at G:C sites on a shuttle vector plasmid replicated in mammalian cells.

Glyoxal is a major product of DNA oxidation in which Fenton-type oxygen free radical-forming systems are involved. To determine the mutation spectrum of glyoxal in mammalian cells and to compare the spectrum with those observed in other experimental systems, we analyzed mutations in a bacterial suppressor tRNA gene (supF) in the shuttle vector plasmid pMY189. We treated pMY189 with glyoxal and immediately transfected it into simian COS-7 cells. The cytotoxicity and mutation frequency increased according to the dose of glyoxal. The majority of glyoxal-induced mutations (48%) were single-base substitutions. Eighty three percent of the single-base substitutions occurred at G:C base pairs. Among them, G:C-->T:A transversions were predominant, followed by G:C-->C:G transversions and G:C-->A:T transitions. A:T-->T:A transversions were also observed. Mutational hotspots within the supF gene were detected. These results suggest that glyoxal may play an important role in mutagenesis induced by oxygen free radicals.     

Singlet Oxygen Induces Predominantly G to T Transversions on a Single-Stranded Shuttle Vector Replicated in Monkey Cells

To elucidate the mechanisms of mutagenesis by singlet oxygen DNA damage in mammalian cells, a SV40-derived single-stranded shuttle vector was exposed to the water soluble endoperoxide 3,3'-(1,4-naphthylidene) dipropionate (NDPO₂). The damaged vector was transfected into monkey COS7 cells and the plasmid progeny exhibited up to 10 fold increase on the mutation frequency in the supF target gene, when compared to untreated vector. The sequence in the supF locus of such mutants revealed that singlet oxygen-induced mutagenesis in single-stranded vector is significantly different from spontaneous mutagenesis. Among the base substitutions, most of the mutations involved deoxyguanosines, being G to T transversions the predominant type of change. The data indicate that mutagenesis by singlet oxygen in mammalian cells may be generated by an error prone bypass of damaged deoxyguanosines at the template DNA.     

DNA sequence context affects UV-induced mutagenesis in Escherichia coli

We investigated the effect of altering the DNA sequence surrounding a mutable target site on the production of ultraviolet light (UV) induced mutations. Site-directed base substitutions were incorporated on both sides of a TAA sequence encoding a UAA nonsense defect in the tyrA14 allele of Escherichia coli. This allele is readily revertable by UV and a total of eight different base substitution mutations can be recovered. Five different strains harboring DNA sequences allowing the formation of 5'-TT, 5'-CT and 5'-TA* photoproducts were constructed and exposed to UV. DNA sequence analysis was used to determine the spectrum of the revertants that were recovered. The results showed that changes at the 3'-base of a TT site were predominantly T to C transitions and T to A transversions. However, unlike the TT site, a 5'-CT site produced a relatively high frequency of T to G transversions. In addition, T to A transversions that could not have been targeted by a cyclobutane-type or [6-4]-type pyrimidine dimer were produced; this result suggested that these mutations may be targeted by a TA* photoproduct. Also, a distinct strand bias was noted for two mechanistically identical base substitutions in a strain having a palindromic target sequence; this result may reflect an unequal damage distribution or processing of photoproducts as a consequence of asymmetric DNA replication. Finally, our results show that DNA sequences expected to allow the greatest density of UV-induced DNA damage produce the highest mutation frequencies. Overall, these findings provide new insights regarding the role of DNA photoproducts in UV mutagenesis.     

Singlet oxygen mutagenicity induced in the lac operon

We have studied the specificity of singlet oxygen (1O2) mutagenesis in single-stranded DNA phage by analysing 1O2-induced mutations in the lac insert of the M13 mp 19 hybrid phage. 107 lac mutants were analysed showing mainly single-base substitutions with a total of 93% and 7% of 40-50 base deletion mutations. Most of the substitutions are G----T and C----A transversions with respectively 27 and 54% of the mutations. The replicative form of the M13 mp 19 DNA (RFDNA) was used as substrate for the 1O2 reactions, there are then two types of progeny phages DNA's. As guanine residues are the targets of the oxidation, it appears that both types of transversions are provided by one type of lesion: the guanine oxidised by 1O2 is read like a thymine by E. coli DNA polymerase-I.     

Strand-specific mutation spectra in repair-proficient and repair-deficient hamster cells

The mutation spectrum induced by UV light has been determined at the hprt locus for both cultured normal (AA8) and UV-sensitive (UV-5) Chinese hamster ovary cells to investigate the effect of DNA repair on the nature of induced mutations. DNA base-pair changes of 23 hprt mutants of AA8 and of 28 hprt mutants of UV-5 were determined by sequence analysis of in vitro amplified hprt cDNA. Almost all mutants in AA8 carried single-base substitutions, transitions and transversions accounting for 38% and 62% of the base changes, respectively. In contrast, in repair-deficient cells (UV-5) tandem and nontandem double mutations represented a considerable portion of the mutations observed (30%), whereas the vast majority of base-pair substitutions were GC greater than AT transitions (87%). Moreover, 5 splice mutants and 2 frameshift mutations were found in the UV-5 collection. In almost all mutants analyzed base changes were located at dipyrimidine sites where UV photoproducts could have been formed. In AA8 the photolesions causing mutations were predominantly located in the nontranscribed strand whereas a strong bias for mutation induction towards photolesions in the transcribed strand was found in UV-5. We hypothesize that preferential removal of lesions from the transcribed strand of the hprt gene accounts for the observed DNA strand specificity of mutations in repair-proficient cells. Furthermore, differences in the degree of misincorporation opposite a lesion for lagging and leading strand DNA synthesis may dictate the pattern of UV-induced mutations in the absence of DNA repair.     

Hydrogen peroxide induces G:C to TA and G:C to C:G transversions in the supF gene of Escherichia coli

A vector plasmid, pZ189, carrying an Escherichia coli supF gene as a target for mutations, was treated with a combination of hydrogen peroxide and Fe³⁺/EDTA complex and propagated in E. coli host cells that had been induced for SOS functions by ultraviolet irradiation. The mutations frequency increased by up to 30-fold over spontaneous background levels with increasing concentrations of hydrogen peroxide. The increase in mutation frequency correlated with an increase in the formation of 8-hydroxydeoxyguanosine in the pZ189 DNA. Sequence analysis of 82 independent supF mutant plasmids revealed that 70 mutants contained base substitutions, with 63 of the 70 involving a G:C base pair, and with G:C→C:G (28 cases) and G:C→T:A (26 cases) transversions predominating. Investigation of the influence of the local DNA sequence on the transversions revealed that the guanine at the center of the triplet 5′-PuGA-3′ was five times more likely to mutate after treatment with hydrogen peroxide than that at the center of 5′PyGN3′. G:C→T:A transversions presumably resulted from mispairing of an altered G (probably 8-hydroxydeoxyguanosine) with deoxyadenosine. The origin of the G:C→C:G transversions may be an as yet unidentified lesion generated by hydrogen peroxide. Mutagenic hotspots for base substitutions were found at positions 133, 160 and 168. Mutation spectra and the positions of mutagenic hotspots, when compared with a previously determined spontaneous mutagenesis spectrum, also provide information on the mechanism of spontaneous mutagenesis.     

Molecular Analysis of Mutations Induced in the Vermilion Gene of Drosophila Melanogaster by Methyl Methanesulfonate

The nature of DNA sequence changes induced by methyl methanesulfonate (MMS) at the vermilion locus of Drosophila melanogaster was determined after exposure of postmeiotic male germ cell stages. MMS is a carcinogen with strong preference for base nitrogen alkylation (s = 0.86). The spectrum of 40 intralocus mutations was dominated by AT----GC transitions (23%), AT----TA transversions (54%) and deletions (14%). The small deletions were preferentially found among mutants isolated in the F1 (8/18), whereas the AT----GC transitions exclusively occurred in the F2 (6/22). The MMS-induced transversions and deletions are presumably caused by N-methyl DNA adducts, which may release apurinic intermediates, known to be a time-related process. Furthermore, MMS produces multilocus deletions, i.e., at least 30% of the F1 mutants analyzed were of this type. A comparison of the mutational spectra of MMS with that produced by ethylnitrosourea (ENU), also in the vermilion locus of Drosophila, reveals major differences: predominantly transition mutations (61% GC----AT and 18% AT----GC) were found in both the F1 and F2 spectrum induced by ENU. It is concluded that the mutational spectrum of MMS is dominated by nitrogen DNA adducts, whereas with ENU DNA sequence changes mainly arose from modified oxygen in DNA.     

Evaluation of the database on mutant frequencies and DNA sequence alterations of vermilion mutations induced in germ cells of Drosophila shows the importance of a neutral mutation detection system

The vermilion gene in Drosophila has extensively been used for the molecular analysis of mutations induced by chemicals in germ cells in vivo. The gene is located on the X-chromosome and is a useful target for the study of mutagenesis since all types of mutations are generated. We have critically evaluated this system with respect to sensitivity for mutation induction and selectivity for different types of mutations, using a database of more than 600 vermilion mutants induced in postmeiotic male germ cells by 18 mutagens. From most of these mutants the mutation has been analysed. These data showed 336 base substitutions, 96 intra-locus DNA rearrangements and 78 multi-locus deletions (MLD). Mutants containing a MLD were either heterozygous sterile or homozygous and hemizygous lethal. The distribution of both basepair (bp) changes and intra-locus rearrangements over the coding region of the vermilion gene was uniform with no preferences concerning 5' or 3' regions, certain exons, splice sites, specific amino acid changes or nonsense mutations. Possible hotspots for base substitutions seem to be related to the type of DNA damage rather than to the vermilion system. Gene mutations other than bp changes were examined on sequence characteristics flanking the deletion breakpoints. Induction frequencies of vermilion mosaic mutants were, in general, higher than those of vermilion complete mutants, suggesting that persistent lesions are the main contributors to the molecular spectra. Comparison of induction frequencies of vermilion mutants and sex-linked recessive lethal (SLRL) mutants for the 18 mutagens showed that the sensitivity of the vermilion gene against a mutagenic insult is representative for genes located on the X-chromosome. The effect of nucleotide excision repair (NER) on the formation of SLRL mutants correlated with an increase of transversions in the vermilion spectra under NER deficient conditions. Furthermore, the clastogenic potency of the mutagens, i.e., the efficiency to induce chromosomal-losses vs. SLRL forward mutations, shows a positive correlation with the percentage of DNA deletions in the molecular spectra of vermilion mutants.     

Excision repair influences the site and strand specificity of sunlight mutagenesis in yeast.

A collection of 384 mutations recovered in a tRNA gene (SUP4-o) following exposure of isogenic excision-repair-proficient (RAD1) or deficient (rad1) strains of the yeast Saccharomyces cerevisiae to sunlight was characterized by DNA sequencing. In each case, greater than 90% of the mutations were single base-pair substitutions with events at G.C pairs constituting most of the changes. However, more than half of these substitutions were transversions in the RAD1 strain whereas transitions predominated in the rad1 strain. Tandem double substitutions were recovered in both strains and the individual changes were exclusively G.C----A.T transitions. The majority of single substitutions, and all tandem double changes, were at base-pairs where the pyrimidine(s) was part of a dipyrimidine sequence and the site specificities were consistent with cyclobutane dimers and/or pyrimidine (6-4) pyrimidone photoproducts contributing to sunlight mutagenesis. Yet, the data also pointed to an important role for lesions that form at G.C pairs and give rise to transversions. Analysis of the strand specificity of sunlight mutagenesis indicated that transitions or transversions at G.C pairs occurred preferentially in SUP4-o at sites where a dipyrimidine or a guanine, respectively, was on the transcribed strand. These biases required a functional excision-repair system.     

Hydroperoxide-induced DNA damage and mutations

Hydroperoxides (ROOH) are believed to play an important role in the generation of free radical damage in biology. Hydrogen peroxide (R=H) is produced by endogenous metabolic and catabolic processes in cells, while alkyl hydroperoxides (R=lipid, protein, DNA) are produced by free radical chain reactions involving molecular oxygen (autooxidation). The role of metal ions in generating DNA damage from hydroperoxides has long been recognized, and several distinct, biologically relevant mechanisms have been identified. Identification of the mechanistic pathways is important since it will largely determine the types of free radicals generated, which will largely determine the spectrum of DNA damage produced. Some mechanistic aspects of the reactions of low valent transition metal ions with ROOH and their role in mutagenesis are reviewed with a perspective on their possible role in the biological generation of DNA damage. A survey of hydroperoxide-induced mutagenesis studies is also presented. In vitro footprinting of DNA damage induced by hydroperoxides provides relevant information on sequence context dependent reactivity, and is valuable for the interpretation of mutation spectra since it represents the damage pattern prior to cellular repair. Efforts in this area are also reviewed.     

Strand specificity for mutations induced by (+)-anti BPDE in the hprt gene in human T-lymphocytes.

Mutations in the hprt gene in T-lymphocyte clones isolated from primary cultures treated with the (+)-anti enantiomer of 7,8-dihydroxy-9,10-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE) in vitro, and from untreated control cultures, were characterized using polymerase chain reaction and direct sequencing of hprt cDNA and genomic fragments. The spectrum of BPDE-induced mutations was very specific and clearly different from the background spectrum, which comprised many different types of mutations. Of the BPDE-induced mutations, 20/22 were transversions of GC base pairs and 18/22 were GC greater than TA transversions, which is in agreement with what has been found in other mammalian systems. While no particular 'hotspot' was observed for BPDE in the hprt gene, a sequence context specificity was detected. Ten of the 14 BPDE-induced mutations in the coding region were located in the sequence context AGG, and 2 in AG dinucleotides, which indicates that such sequences are sensitive to BPDE mutagenesis. Nine of the 22 BPDE-induced mutations and 2/12 background point mutations caused mRNA splicing errors. Six of the BPDE-induced splicing errors were caused by GC greater than TA transversions in the AG dinucleotide of different splice acceptor sites, which indicates that these sites may be frequent targets of BPDE mutagenesis. All mutated GC base pairs in the BPDE-induced spectrum were oriented so that the guanine was located on the non-transcribed strand. Assuming that the premutagenic lesion in these cases was covalent binding of BPDE to guanine and that BPDE bound randomly to both strands, the strand specificity of the BPDE-induced mutations indicates that preferential excision repair of BPDE adducts on the transcribed strand occurs in the hprt gene in human T-cells.     

DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli

The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but -2 frameshifts and +1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G.C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-2-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.     

DNA repair and sequence context affect 1O2-induced mutagenesis in bacteria

Electronic excited molecular oxygen (singlet oxygen, ¹O₂) is known to damage DNA, yielding mutations. In this work, the mutagenicity induced by ¹O₂ in a defined sequence of DNA was investigated after replication in Escherichia coli mutants deficient for nucleotide and base excision DNA repair pathways. For this purpose a plasmid containing a ¹O₂-damaged 14 base oligonucleotide was introduced into E.coli by transfection and mutations were screened by hybridization with an oligonucleotide with the original sequence. Mutagenesis was observed in all strains tested, but it was especially high in the BH20 (fpg), AYM57 (fpg mutY) and AYM84 (fpg mutY uvrC) strains. The frequency of mutants in the fpg mutY strain was higher than in the triple mutant fpg mutY uvrC, suggesting that activity of the UvrABC excinuclease can favor the mutagenesis of these lesions. Additionally, most of the mutations were G→T and G→C transversions, but this was dependent on the position of the guanine in the sequence and on repair deficiency in the host bacteria. Thus, the kind of repair and the mutagenesis associated with ¹O₂-induced DNA damage are linked to the context of the damaged sequence.     

Singlet oxygen induced mutation spectrum in mammalian cells.

In order to characterize the molecular nature of singlet oxygen (1O2) induced mutations in mammalian cells, a SV40-based shuttle vector (pi SVPC13) was treated with singlet oxygen arising from the thermal decomposition of the water-soluble endoperoxide of 3,3'-(1,4-naphthylidene) dipropionate (NDPO2). After the passage of damaged plasmid through monkey COS7 cells, the vector was shuffled into E. coli cells, allowing the screening of supF mutants. The mutation spectrum analysis shows that single and multiple base substitutions arose in 82.5% of the mutants, the others being rearrangements. The distribution of mutations within the supF gene is not random and some hotspots are evident. Most of the point mutations (98.4%) involve G:C base pairs and G:C to T:A transversion was the most frequent mutation (50.8%), followed by G:C to C:G transversion (32.8%). These results indicate that mutagenesis in mammalian cells, mediated by 1O2-induced DNA damage, is targeted selectively at guanine residues.     

Induction of transversion mutations in Escherichia coli by N-methyl-N''-nitro-N-nitrosoguanidine is SOS dependent.

Escherichia coli alkA mutants, which are deficient for an inducible DNA glycosylase, 3-methyladenine-DNA glycosylase II, are sensitive to mutagenesis by low doses of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). As many as 90% of the alkA-dependent mutations induced by MNNG are also umuC+ dependent and thus are due to DNA lesions that are substrates for the mutagenic functions of the SOS response. A great number of these mutations are base substitutions at A . T sites, particularly A . T transversions. We discuss which DNA lesions may be responsible for these mutations. Our results show that the induction of 3-methyladenine-DNA glycosylase II, which occurs as part of the adaptive response to alkylating agents such as MNNG, significantly reduces the mutagenicity as well as the lethality of alkylation damage.     

Detection of tandem CC-->TT mutations induced by oxygen radicals using mutation-specific PCR.

DNA lesions caused by reactive oxygen species (ROS) are considered to be one of the major contributors to DNA damage and mutagenesis. In this study, we developed a modification of allele-specific PCR to detect CC-->TT mutations caused by oxidative damage. These tandem mutations have been previously demonstrated to be indicative of oxygen damage in the absence of UV-irradiation. Using a CC target site in the rat DNA polymerase beta (pol beta) gene and a thermostable restriction enzyme that cuts the wild type sequence but not the TT mutation, we demonstrate that the TT mutation can be preferentially amplified from plasmid DNA damaged by oxygen radicals but not other DNA-damaging agents. We evaluated the potential utility of this assay in screening for mutations in cells and in analyzing those that arise during clonal proliferation in carcinogenesis.     

UV-induced hprt mutations in a UV-sensitive hamster cell line from complementation group 3 are biased towards the transcribed strand.

The molecular nature of 254 nm ultraviolet light (UV)-induced mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in UV24 Chinese hamster ovary (CHO) cells, which are defective in nucleotide excision repair, was determined. Sequence analysis of 19 hprt mutants showed that single base substitutions (9 mutants) and tandem base changes (7 mutants) dominated the UV mutation spectrum in this cell line. Sixty-five percent of the base substitutions were GC greater than AT transitions, whereas the rest consisted of transitions and transversions at AT base pairs. Analysis of the distribution of dipyrimidine sites over the two DNA strands, where the photoproducts causing these mutations presumably were formed, showed that 12 out of 14 mutations were located in the transcribed strand of the hprt gene. A similar strand distribution of mutagenic photoproducts as in UV24 has previously been found in two other UV-sensitive Chinese hamster cell lines (V-H1 and UV5), indicating that under defective nucleotide excision repair conditions the induction of mutations is strongly biased towards lesions in the transcribed strand of the hprt gene. A plausible explanation for this phenomenon is that during DNA replication large differences exist in the error rate with which DNA polymerase(s) bypass lesions in the templates for the leading and lagging strand, respectively.     

Novel point mutations in mitochondrial 16S rRNA gene of Chinese hamster cells

To know the nature and mechanisms of spontaneous mutations in mitochondrial DNA (mtDNA), we determined, by direct cycle sequencing, the nucleotide sequence of the 3' terminal region of the mitochondrial 16S rRNA gene from chloramphenicol-resistant (CAP-R) mutants isolated in Chinese hamster V79 cells. Four different base substitutions were identified in common for the six CAP-R mutants. All mutations were heteroplasmic. One A to G transition was mapped at a site within the putative peptidyl transferase domain, the target region for chloramphenicol, and one G to A transition and two T to G transversions were located within the two different segments which form the stems of the hairpin loop structures attached to this key domain in the predicted secondary structure of 16S rRNA. The mutations detected in this study do not map to the same sites where CAP-R mutations were found previously in mammalian cells. Allele specific-PCR analyses revealed that all four mutations occurred on a single mutant-DNA molecule, but not on several ones independently. Together with the other previous reports, our data suggest that spontaneous mtDNA mutations may not be caused exclusively by oxidative DNA damage at least in 16S rRNA gene.