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.     

Characterization of mutational specificity within the lacI gene for a mutD5 mutator strain of Escherichia coli defective in 3''----5'' exonuclease (proofreading) activity.

The mutD (dnaQ) gene of Escherichia coli codes for the epsilon subunit of the DNA polymerase III holoenzyme which is involved in 3'----5' exonuclease proofreading activity. We determined the mutational specificity of the mutator allele, mutD5, in the lacI gene of E. coli. The mutD5 mutation preferentially produces single base substitutions as judged from the enhanced fraction of lacI nonsense mutations and the spectrum of sequenced dominant lacI (lacId) and constitutive lacO (lacOc) mutations which were predominantly (69/71) single nucleotide substitutions. The distribution of amber lacI and sequenced lacId mutations revealed that transitions occur more frequently than transversions. A . T----G . C and G . C----A . T transitions were equally frequent and, with one major exception, evenly distributed among numerous sites. Among the transversions, A . T----T . A events were the most common, A . T----C . G substitutions were rare, and G . C----C . G changes were not detected. Transversions were unequally distributed among a limited number of sites with obvious hotspots. All 11 sequenced transversions had a consensus neighboring sequence of 5'-C-C-(mutated G or A)-C-3'. Although no large deletions or complex mutational events were recovered, sequencing revealed that mutD5 induced single nucleotide deletions within consecutive G X C sequences. An extraordinary A . T----G . C transition hotspot occurred at nucleotide position +6 in the lac operator region; the mutD5 mutation frequency of this single base pair was calculated to be 1.2 X 10(-3).     

Specificity of mutational DNA sequence changes induced by X-rays in the cloned Escherichia coli crp gene.

Plasmid DNA carrying the adenosine 3',5'-cyclic monophosphate receptor protein (crp) gene of Escherichia coli was irradiated, in solution, with X-rays, and the mutations produced in the crp gene were assayed by transforming the recipient E. coli cells. Ninety-six mutant clones were isolated, and mutational changes were determined by DNA sequencing. Of the 92 mutations thus detected, 74 represented base substitution mutations and the remaining 18 were frameshifts. The base substitutions included 56 G:C to A:T transitions, 10 G:C to T:A transversions and 7 G:C to C:G transversions. An A:T to G:C transition was found only once, and neither an A:T to T:A nor an A:T to C:G transversion was detected. The frameshift mutations consisted of 11 one-base deletions and 7 one-base insertions. Accordingly, G:C to A:T transition was the predominant type of mutation, which constituted 76% (56/74) of the total base substitutions and 60% (56/92) of all detected mutations. Furthermore, of the 56 transitions, about three-quarters (41 clones) clustered at an identical site, a cytosine residue at the 706 position, demonstrating that this site is a distinct hot spot for X-ray mutagenesis. These results raise the possibility that radiation-induced mutations may not necessarily occur randomly, at least in certain cases.     

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.     

Reduction of ENU-induced transversion mutations by the isoflavone genistein in Escherichia coli

In studies of mutagenesis induced by the carcinogen N-ethyl-N-nitrosourea (ENU) in the bacterium Escherichia coli FX-11, it was observed that G:C to A:T transitions did not require the inducible umuDC gene products, while a portion of the A:T to G:C transitions and all transversion mutations were dependent on a functional umuC gene. This observation suggested that the different base substitutions may result from differential processing of specific DNA adducts produced by ENU. To further understand these processes, we have investigated the effect of the soybean isoflavone genistein on the production of ENU-induced mutations. This compound, in particular, has been shown to exhibit numerous effects including the inhibition of the growth or proliferation of a variety of cancers, inhibition of angiogenesis, inhibition of tyrosine protein kinases and anti-oxidant properties. In our experiments, tyrosine defective (TyrA(-)) E. coli were exposed to ENU and a portion of the ENU-treated cells were exposed to genistein. The results showed a three-fold reduction in the overall mutation frequency when cells were treated with genistein subsequent to ENU-exposure and this anti-mutagenic effect was dependent on the dose of genistein employed. However, only certain types of base substitution mutagenesis were affected. In particular, transversion mutations were reduced an average of about 8.5-fold, while transitions were not greatly affected. In addition, UV-mutagenesis was reduced about three-fold and induction of the SOS response (as monitored with a sulA-lacZ fusion) was decreased. These results suggest that genistein may interfere with expression of the SOS response, including the UmuC-mediated lesion bypass mechanism that is necessary for UV-mutagenesis and the generation of transversions by ENU in E. coli.     

The effect of folate deficiency on the hprt mutational spectrum in Chinese hamster ovary cells treated with monofunctional alkylating agents.

Folic acid deficiency acts synergistically with alkylating agents to increase DNA strand breaks and mutant frequency at the hprt locus in Chinese hamster ovary (CHO) cells. To elucidate the mechanism of this synergy, molecular analyses of hprt mutants were performed. Recently, our laboratory showed that folate deficiency increased the percentage of clones with intragenic deletions after exposure to ethyl methanesulfonate (EMS) but not N-nitroso-N-ethylurea (ENU) compared to clones recovered from folate replete medium. This report describes molecular analyses of the 37 hprt mutant clones obtained that did not contain deletions. Folate deficient cells treated with EMS had a high frequency of G>A transitions at non-CpG sites on the non-transcribed strand, particularly when these bases were flanked on both sides by G:C base pairs. Thirty-three percent of these mutations were in the run of six G's in exon 3. EMS-treated folate replete cells had a slightly (but not significantly) lower percentage of G>A transitions, and the same sequence specificity. Treatment of folate deficient CHO cells with ENU resulted in predominantly T>A transversions and C>T transitions relative to the non-transcribed strand. These findings suggest a model to explain the synergy between folate deficiency and alkylating agents: (1) folate deficiency causes extensive uracil incorporation into DNA; (2) greatly increased utilization of base excision repair to remove uracil and to correct alkylator damage leads to error-prone DNA repair. In the case of EMS, this results in more intragenic deletions and G:C to A:T mutations due to impaired ligation of single-strand breaks generated during base excision repair and a decreased capacity to remove O6-ethylguanine. In the case of ENU additional T>A transversions and C>T transitions are seen, perhaps due to mis-pairing of O2-ethylpyrimidines. Correction of folate deficiency may reduce the frequency of these types of genetic damage during alkylator therapy.     

DNA base changes and RNA levels in N-acetoxy-2-acetylaminofluorene-induced dihydrofolate reductase mutants of Chinese hamster ovary cells

Formerly, we isolated a series of dihydrofolate reductase-deficient Chinese hamster ovary cell mutants that were induced by N-acetoxy-2-acetylaminofluorene. Deletions and complex gene rearrangements were detected in 28% of these mutants; 72% contained putative point mutations. In the present study, we have localized the putative point mutations in the 25,000 base dhfr gene by RNase heteroduplex mapping. Assignment of a position for each mutation was successful in 16 of 19 mutants studied. We cloned DNA fragments containing the mapped mutations from nine mutants into a bacteriophage lambda vector. In the case of 11 other mutants, DNA was amplified by the polymerase chain reaction procedure. Sequence analysis of cloned and amplified DNA confirmed the presence of point mutations. Most mutants (90%) carried base substitutions; the rest contained frameshift mutations. Of the point mutations, 75% were G.C to T.A transversions in either the dhfr coding sequence or at splice sites; transition G.C to A.T mutations were found in two mutants (10%). In one of these transition mutants, the base substitution occurred at the fifth base of the third intron. Of the frameshift mutations, one was a deletion of G.C pair and the other was an insertion of an A.T pair. Of the mapped mutants, 38% exhibited greatly reduced (approximately 10-fold) steady-state levels of dhfr mRNA. All eight sequenced mutants displaying this phenotype contained premature chain termination codons. Normal levels of dhfr mRNA were observed in five missense mutants and in five mutants carrying nonsense codons in the translated portion of exon VI. Taken together with the results of other mutagens at this locus, we conclude that the low dhfr mRNA phenotype is correlated with the presence of nonsense codons in exons II to V but not in the last exon of the dhfr gene.     

Differentiation between Amber and Ochre Mutants of Yeast by Reversion with 4-Nitroquinoline-1-Oxide

4-nitroquinoline-1-oxide (NQO) induces high frequencies of intragenic revertants of amber (UAG) but not ochre (UAA) mutants of yeast. Distinction of the amber and ochre codons was made with well-characterized nonsense mutants of the iso-1-cytochrome c gene (cyc1 mutants) as well as with nonsense mutants having nutritional requirements. Thus the NQO-induced reversion frequencies corroborated the assignments that were based on the pattern of amino acid replacements in intragenic revertants and on the speficity of suppression. It was concluded from these results and from the results of a previous investigation with other cyc1 mutants (Prakash, Stewart and Sherman 1974) that NQO induces transversions of G:C base pairs at many sites and that the specificity is not strongly influenced by neighboring base pairs in at least the strains examined in these studies. NQO was previously shown to induce G:C → A:T transitions at least at one site and this and the previous study established that it does not significantly mutate A:T base pairs at numerous sites. Thus NQO can be used to selectively mutate G:C base pairs and to determine if the pathways of reverse mutations involve G:C base pairs. Suppressors that act on either amber or ochre mutants were induced with NQO, indicating that they can arise by mutations of G:C base pairs.     

Characterization of TCHQ-induced genotoxicity and mutagenesis using the pSP189 shuttle vector in mammalian cells

Tetrachlorohydroquinone (TCHQ) is a major toxic metabolite of the widely used wood preservative, pentachlorophenol (PCP), and it has also been implicated in PCP genotoxicity. However, the underlying mechanisms of genotoxicity and mutagenesis induced by TCHQ remain unclear. In this study, we examined the genotoxicity of TCHQ by using comet assays to detect DNA breakage and formation of TCHQ-DNA adducts. Then, we further verified the levels of mutagenesis by using the pSP189 shuttle vector in A549 human lung carcinoma cells. We demonstrated that TCHQ causes significant genotoxicity by inducing DNA breakage and forming DNA adducts. Additionally, DNA sequence analysis of the TCHQ-induced mutations revealed that 85.36% were single base substitutions, 9.76% were single base insertions, and 4.88% were large fragment deletions. More than 80% of the base substitutions occurred at G:C base pairs, and the mutations were G:C to C:G, G:C to T:A or G:C to A:T transversions and transitions. The most common types of mutations in A549 cells were G:C to A:T (37.14%) and A:T to C:G transitions (14.29%) and G:C to C:G (34.29%) and G:C to T:A (11.43%) transversions. We identified hotspots at nucleotides 129, 141, and 155 in the supF gene of plasmid pSP189. These mutation hotspots accounted for 63% of all single base substitutions. We conclude that TCHQ induces sequence-specific DNA mutations at high frequencies. Therefore, the safety of using this product would be carefully examined.     

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.     

Cr(III)-mediated crosslinks of glutathione or amino acids to the DNA phosphate backbone are mutagenic in human cells.

Carcinogenic Cr(VI) compounds were previously found to induce amino acid/glutathione-Cr(III)-DNA crosslinks with the site of adduction on the phosphate backbone. Utilizing the pSP189 shuttle vector plasmid we found that these ternary DNA adducts were mutagenic in human fibroblasts. The Cr(III)-glutathione adduct was the most potent in this assay, followed by Cr(III)-His and Cr(III)-Cys adducts. Binary Cr(III)-DNA complexes were only weakly mutagenic, inducing a significant response only at a 10 times higher number of adducts compared with Cr(III)-glutathione. Single base substitutions at the G:C base pairs were the predominant type of mutations for all Cr(III) adducts. Cr(III), Cr(III)-Cys and Cr(III)-His adducts induced G:C-->A:T transitions and G:C-->T:A transversions with almost equal frequency, whereas the Cr(III)-glutathione mutational spectrum was dominated by G:C-->T:A transversions. Adduct-induced mutations were targeted toward G:C base pairs with either A or G in the 3' position to the mutated G, while spontaneous mutations occurred mostly at G:C base pairs with a 3' A. No correlation was found between the sites of DNA adduction and positions of base substitution, as adducts were formed randomly on DNA with no base specificity. The observed mutagenicity of Cr(III)-induced phosphotriesters demonstrates the importance of a Cr(III)-dependent pathway in Cr(VI) carcinogenicity.     

Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Mutagenesis of the N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine DNA adduct in mammalian cells. Sequence context effects.

Site-specifically modified oligodeoxynucleotides were used to investigate the mutagenic properties of a major cooked food mutagen-derived DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (dG-C8-PhIP). dG-C8-PhIP-modified oligodeoxynucleotides were prepared by reacting an oligodeoxynucleotide containing a single dG (5'-TCCTCCTXGCCTCTC, where X = C, A, G, or T) with N-acetoxy-PhIP. The unmodified and dG-C8-PhIP-modified oligomers were inserted into single-stranded phagemid vectors. These single-stranded vectors were transfected into simian kidney (COS-7) cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. When dC was at the 5'-flanking position to dG-C8-PhIP, preferential incorporation of dCMP, the correct base, was observed opposite the dG-C8-PhIP. Targeted G --> T transversions were detected, along with lesser amounts of G --> A transitions and G --> C transversions. No mutations were detected for the unmodified vector. The influence of sequence context on the dG-C8-PhIP mutation frequency and spectrum was also explored. When the dC 5'-flanking base was replaced by dT, dA, or dG, the mutational spectra were similar to that observed with dC-flanking base. Higher mutational frequencies (28-30%) were observed when dC or dG was 5' to dG-C8-PhIP. A lower mutational frequency (13%) was observed when dA was at the 5' to the lesion. Single-base deletions were detected only when dG or dT flanked the adduct. We conclude that dG-C8-PhIP is mutagenic, generating primarily G --> T transversions in mammalian cells. The mutational frequency and specificity of dG-C8-PhIP vary depending on the neighboring sequence context.     

DNA alkylation repair limits spontaneous base substitution mutations in Escherichia coli.

The Escherichia coli Ada and Ogt DNA methyltransferases (MTases) are known to transfer simple alkyl groups from O6-alkylguanine and O4-alkylthymine, directly restoring these alkylated DNA lesions to guanine and thymine. In addition to being exquisitely sensitive to the mutagenic effects of methylating agents, E. coli ada ogt null mutants display a higher spontaneous mutation rate than the wild type. Here, we determined which base substitution mutations are elevated in the MTase-deficient cells by monitoring the reversion of six mutated lacZ alleles that revert via each of the six possible base substitution mutations. During exponential growth, the spontaneous rate of G:C to A:T transitions and G:C to C:G transversions was elevated about fourfold in ada ogt double mutant versus wild-type E. coli. Furthermore, compared with the wild type, stationary populations of the MTase-deficient E. coli (under lactose selection) displayed increased G:C to A:T and A:T to G:C transitions (10- and 3-fold, respectively) and increased G:C to C:G, A:T to C:G, and A:T to T:A transversions (10-, 2.5-, and 1.7-fold, respectively). ada and ogt single mutants did not suffer elevated spontaneous mutation rates for any base substitution event, and the cloned ada and ogt genes each restored wild-type spontaneous mutation rates to the ada ogt MTase-deficient strains. We infer that both the Ada MTase and the Ogt MTase can repair the endogenously produced DNA lesions responsible for each of the five base substitution events that are elevated in MTase-deficient cells. Simple methylating and ethylating agents induced G:C to A:T and A:T to G:C transitions in these strains but did not significantly induce G:C to C:G, A:T to C:G, and A:T to T:A transversions. We deduce that S-adenosylmethionine (known to e a weak methylating agent) is not the only metabolite responsible for endogenous DNA alkylation and that at least some of the endogenous metabolites that cause O-alkyl DNA damage in E. coli are not simple methylating or ethylating agents.     

The importance of distinct metabolites of N-nitrosodiethylamine for its in vivo mutagenic specificity

Although N-nitrosodiethylamine (NDEA) is a potent carcinogen in rodents and a probable human carcinogen, little attempts were made to characterize its mutation spectrum in higher eukaryotes. We have compared forward mutation frequencies at multiple (700) loci with the mutational spectrum induced at the vermilion gene of Drosophila, after exposure of post- and pre-meiotic male germ cells to NDEA. Among 30 vermilion mutants collected from post-meiotic stages were 12 G:C-->A:T transitions (40%), 8 A:T-->T:A transversions (27%), and 4 structural rearrangements (13%). The remainder were three A:T-->G:C transitions, two G:C-->C:G transversions and one G:C-->T:A transversion. The results show that although NDEA induces predominantly transitions (40% G:C-->A:T and 10% A:T-->G:C), the frequencies of transversions (37%, of which 27% of A:T-->T:A transversions) and especially of rearrangements (13%) are remarkably high. This mutation spectrum differs significantly from that produced by the direct-ethylating agent N-ethylnitrosourea (ENU), although the relative distribution of ethylated DNA adducts is similar for both carcinogens. These differences, in particular the occurrence of rearrangements, are most likely the result of the requirement of NDEA for bioactivation. Since all four rearrangements were collected from non-metabolizing spermatozoa (or late spermatids), it is hypothesized that they derived from acetaldehyde, a stable metabolite of NDEA. Due to its cytotoxicity, attempts to isolate vermilion mutants from NDEA-exposed pre-meiotic cells were largely unsuccessful, because only two mutants (one A:T-->G:C transition and one 1bp insertion) were collected from those stages. Our results show that NDEA is capable of generating carcinogenic lesions other than base pair substitutions.     

The Influence of Local DNA Sequence and DNA Repair Background on the Mutational Specificity of 1-Nitroso-8-Nitropyrene in Escherichia Coli: Inferences for Mutagenic Mechanisms

We have examined the mutational specificity of 1-nitroso-8-nitropyrene (1,8-NONP), an activated metabolite of the carcinogen 1,8-dinitropyrene, in the lacI gene of Escherichia coli strains which differ with respect to nucleotide excision repair (+/- delta uvrB) and MucA/B-mediated error-prone translesion synthesis (+/- pKM101). Several different classes of mutation were recovered, of which frameshifts, base substitutions, and deletions were clearly induced by 1,8-NONP treatment. The high proportion of point mutations (> 92%) which occurred at G.C sites correlates with the percentage of 1,8-NONP-DNA adducts which occur at the C(8) position of guanine. The most prominent frameshift mutations were -(G.C) events, which were induced by 1,8-NONP treatment in all strains, occurred preferentially in runs of guanine residues, and whose frequency increased markedly with the length of the reiterated sequence. Of the base substitution mutations G.C-->T.A transversions were induced to the greatest extent by 1,8-NONP. The distribution of the G.C-->T.A transversions was not influenced by the nature of flanking bases, nor was there a strand preference for these events. The presence of plasmid pKM101 specifically increased the frequency of G.C-->T.A transversions by a factor of 30-60. In contrast, the -(G.C) frameshift mutation frequency was increased only 2-4-fold in strains harboring pKM101 as compared to strains lacking this plasmid. There was, however, a marked influence of pKM101 on the strand specificity of frameshift mutation; a preference was observed for -G events on the transcribed strand. The ability of the bacteria to carry out nucleotide excision repair had a strong effect on the frequency of all classes of mutation but did not significantly influence either the overall distribution of mutational classes or the strand specificity of G.C-->T.A transversions and -(G.C) frameshifts. Deletion mutations were induced in the delta uvr, pKM101 strain. The endpoints of the majority of the deletion mutations were G.C rich and contained regions of considerable homology. The specificity of 1,8-NONP-induced mutation suggests that DNA containing 1,8-NONP adducts can be processed through different mutational pathways depending on the DNA sequence context of the adduct and the DNA repair background of the cell.     

Mutagenic specificity of N-acetoxy-3-aminobenzanthrone, a major metabolically activated form of 3-nitrobenzanthrone, in shuttle vector plasmids propagated in human cells

3-Nitrobenzanthrone (3-NBA) is a potent environmental mutagen and a potential human carcinogen present in diesel exhaust and airborne particulates. N-acetoxy-3-aminobenzanthrone (N-Aco-ABA) has been shown to be a major reactive metabolite of 3-NBA, which mainly produces adducts with guanine and adenine in cellular DNA. Here we analyzed mutations induced by N-Aco-ABA using supF shuttle vector plasmids to elucidate the mutagenic specificity of 3-NBA in human cells. Base sequence analysis of more than 100 plasmids with supF mutations induced in wildtype and DNA repair-deficient XP cells revealed that the major mutation was base substitutions of which the majority (42 and 38%, respectively) were G:C to T:A transversions. The next major mutation was G:C to A:T and A:T to G:C base substitutions in wildtype and XP cells, respectively. The DNA polymerase stop assay using N-Aco-ABA-treated plasmids as a template showed that most stop signals, i.e., adducted sites, appeared at G:C sites. These results suggest that N-Aco-ABA binds preferably to guanine rather than adenine, and adducted adenine is repaired more efficiently by the nucleotide excision repair. Error-prone DNA polymerases could insert adenine at sites opposite to N-Aco-ABA-adducted guanine, which leads to G:C to T:A transversion. These findings could be very important to evaluate the human lung cancer risk of environmental 3-NBA.     

Further studies with a cell immortalization assay to investigate the mutation signature of aristolochic acid in human p53 sequences

To test hypotheses on the origins of p53 mutations in human tumors, novel strategies are needed for generating mutation spectra experimentally. To this end we developed an assay employing Hupki (Human p53 knock-in) mouse embryonic fibroblasts (HUFs). Here we examine p53 mutations induced by aristolochic acid I (AAI)), the carcinogen probably responsible for Chinese herbal nephropathy. Six immortalized cultures (cell lines) from 18 HUF primary cultures exposed at passage 1 for 48 h to 50 microM AAI harbored p53 mutations in the human DNA binding domain sequence of the Hupki p53 tumor suppressor gene. The most frequently observed mutation was A to T transversion, corroborating our previous mutation study with AAI, and consistent with the presence of persistent AAI-adenine adducts found both in DNA of exposed patients and in DNA of AAI-exposed HUF cells. One of the mutations was identical in position (codon 139) and base change (A to T on the non-transcribed strand) to the single p53 mutation that has thus far been characterized in a urothelial tumor of a nephropathy patient with documented AAI exposure. Of the seven p53 mutations identified thus far in >60 HUF cell lines that immortalized spontaneously (no carcinogen treatment), none were A:T to T:A transversions. In addition, no A to T substitutions were identified among the previously reported set of 18 mutations in HUF cell lines derived from B(a)P treatment in which transversions at G:C base pairs predominated.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

Delayed transfection of DNA after riboflavin mediated photosensitization increases G:C to C:G transversions of supF gene in Escherichia coli mutY strain.

We have previously reported that the majority of base substitution mutations of the Escherichia coli supF gene induced by riboflavin mediated photosensitization were G:C to C:G changes, in addition to G:C to T:A changes which were probably caused by 8-hydroxyguanine (oh(8)Gua), in wild type and mutM mutator mutant strains. This implies that lesions other than oh(8)Gua are produced by riboflavin-photosensitization. G:C to C:G base substitutions have been found in the mutations induced by ionizing radiation and reactive oxygen species, as well as spontaneous mutation. To characterize the G:C to C:G mutation, riboflavin- photosensitized plasmid DNA carrying the supF gene was left at room temperature for 5 h in the dark before transfection. The delayed transfection gave a mutational spectrum different from that for immediate transfection. G:C to C:G transversions significantly increased in mutY mutator strain, in which the transversion was not detected in the immediate transfection. Lesions causing G:C to C:G changes increased during 5-h holding after photosensitization and MutY protein presumably takes part in this type of base change mutation.