Specificity of Base Substitutions Induced by the Acridine Mutagen Icr-191: Mispairing by Guanine N7 Adducts as a Mutagenic Mechanism

As the most nucleophilic site in DNA, the guanine N7 atom is a major site of adduction by a large number of alkylating mutagens and carcinogens. Aflatoxin B1, a powerful mutagen, is believed to act through its reaction with this DNA site. On the basis of the specificity of base substitutions induced by various adduct forms of aflatoxin, we have proposed that bulky guanine N7 adducts elicit base substitutions by two mechanisms. The first mechanism is similar to that observed for a number of bulky noninstructive lesions, whereas the second mechanism invokes mispairing between N7-adducted guanine and thymine. A prediction of the mispairing hypothesis is that diverse bulky guanine N7 adducts (regardless of structural similarities with the aflatoxins) should induce predominantly G-to-A transitions. Accordingly, we have recently observed that base substitutions induced by the acridine half-mustard ICR-191 in the M13 double-stranded DNA transfection system are predominantly G:C-to-A:T transitions. Here, by transfecting ICR-191-treated M13 AB28 single-stranded DNA into Escherichia coli, we show that base substitutions are predominantly targeted to guanines. Since the N7-adducted-guanine:thymine mispairing is proposed to require N1 deprotonation promoted by the primary N7 lesion, guanine imidazole ring-opening should abolish this mispairing property, and thereby alter the specificity of mutagenesis. Here, we show that the incubation of ICR-191-treated RF DNA at pH 10.5 results in a significant reversal of the specificity of G:C-targeted substitutions such that G-to-T transversions predominated over G-to-A transitions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evading the proofreading machinery of a replicative DNA polymerase: induction of a mutation by an environmental carcinogen

DNA replication fidelity is dictated by DNA polymerase enzymes and associated proteins. When the template DNA is damaged by a carcinogen, the fidelity of DNA replication is sometimes compromized, allowing mispaired bases to persist and be incorporated into the DNA, resulting in a mutation. A key question in chemical carcinogenesis by metabolically activated polycyclic aromatic hydrocarbons (PAHs) is the nature of the interactions between the carcinogen-damaged DNA and the replicating polymerase protein that permits the mutagenic misincorporation to occur. PAHs are environmental carcinogens that, upon metabolic activation, can react with DNA to form bulky covalently linked combination molecules known as carcinogen-DNA adducts. Benzo[a]pyrene (BP) is a common PAH found in a wide range of material ingested by humans, including cigarette smoke, car exhaust, broiled meats and fish, and as a contaminant in other foods. BP is metabolically activated into several highly reactive intermediates, including the highly tumorigenic (+)-anti-benzo[a]pyrene diol epoxide (BPDE). The primary product of the reaction of (+)-anti-BPDE with DNA, the (+)-trans-anti-benzo[a]pyrene diol epoxide-N(2)-dG ((+)-ta-[BP]G) adduct, is the most mutagenic BP adduct in mammalian systems and primarily causes G-to-T transversion mutations, resulting from the mismatch of adenine with BP-damaged guanine during replication. In order to elucidate the structural characteristics and interactions between the DNA polymerase and carcinogen-damaged DNA that allow a misincorporation opposite a DNA lesion, we have modeled a (+)-ta-[BP]G adduct at a primer-template junction within the replicative phage T7 DNA polymerase containing an incoming dATP, the nucleotide most commonly mismatched with the (+)-ta-[BP]G adduct during replication. A one nanosecond molecular dynamics simulation, using AMBER 5.0, has been carried out, and the resultant trajectory analyzed. The modeling and simulation have revealed that a (+)-ta-[BP]G:A mismatch can be accommodated stably in the active site so that the fidelity mechanisms of the polymerase are evaded and the polymerase accepts the incoming mutagenic base. In this structure, the modified guanine base is in the syn conformation, with the BP moiety positioned in the major groove, without interfering with the normal protein-DNA interactions required for faithful polymerase function. This structure is stabilized by a hydrogen bond between the modified guanine base and dATP partner, hydrophobic interactions between the BP moiety and the polymerase, a hydrogen bond between the modified guanine base and the polymerase, and several hydrogen bonds between the BP moiety and polymerase side-chains. Moreover, the G:A mismatch in this system closely resembles the size and shape of a normal Watson-Crick pair. These features reveal how the polymerase proofreading machinery may be evaded in the presence of a mutagenic carcinogen-damaged DNA, so that a mismatch can be accommodated readily, allowing bypass of the adduct by the replicative T7 DNA polymerase.     

Induction of G.C to A.T transitions by the acridine half-mustard ICR-191 supports a mispairing mechanism for mutagenesis by some bulky mutagens

As the most nucleophilic atom in DNA, the guanine N7 atom is a major site of attack for a large number of chemical mutagens as well as chemotherapeutic agents. Paradoxically, while methylation of guanine N7 is believed to be largely nonmutagenic, aflatoxin B1, among the most potent mutagens, appears to exert its mutagenic activity through adduction at this site. On the basis of an analysis of the specificity of mutations induced by various adduct forms of aflatoxin B1, we have previously proposed mechanisms that can both resolve the paradox and account for the specificity of mutagenesis by aflatoxin B1. The hypothesized mechanisms specify how a bulky guanine N7 lesion can promote G.C to A.T transitions as well as frame-shift mutations. Since the proposed mechanisms are in principle lesion-independent, a simple test of the proposed mechanisms would be to examine the specificity of mutations induced by a structurally different bulky guanine N7 adduct. Toward this goal, M13 replicative form DNA was subjected to in vitro adduction with the acridine mutagen ICR-191 and transfected into Escherichia coli. Mutations in the LacZ(alpha) gene segments were scored and defined at the sequence level. The results show that ICR-191 adduction induces both base substitutions and frame shifts with near-equal efficiency. A clear majority of base substitutions were G.C to A.T transitions. On the other hand, unlike aflatoxin B1 which could induce both -1 and +1 frameshifts, ICR-191 appears to predominantly induce +1 frame shifts. This preference appears to arise by lesion-dependent mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)     

trans-Lesion synthesis past bulky benzo[a]pyrene diol epoxide N2-dG and N6-dA lesions catalyzed by DNA bypass polymerases

The effectiveness of in vitro primer elongation reactions catalyzed by human bypass DNA polymerases kappa (hDinB1), pol eta (hRad30A), pol iota (hRad30B), and yeast pol zeta (Rev3 and Rev7) in site-specifically modified template oligonucleotide strands were studied in vitro. The templates contained single bulky lesions derived from the trans-addition of the mutagenic (+)- or (-)-enantiomers of r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (a metabolite of the environmental carcinogen benzo[a]pyrene), to the exocyclic amino groups of guanine or adenine in oligonucleotide templates 33, or more, bases long. In "running start" primer extension reactions, pol kappa effectively bypassed both the stereoisomeric (+)- and (-)-trans-guanine adducts but not the analogous adenine adducts. In sharp contrast, pol eta, which exhibits considerable sequence homology with pol kappa (both belong to the group of Y family polymerases), is partially blocked by the guanine adducts and the (-)-trans-adenine adduct, although the stereoisomeric (+)-trans-adenine adduct is more successfully bypassed. Neither pol iota nor pol zeta, either alone or in combination, were effective in trans-lesion synthesis past the same adducts. In all cases, the fidelity of insertion is dependent on adduct stereochemistry and structure. Generally, error-free nucleotide insertion opposite the lesions tends to depend more on adduct stereochemistry than error-prone insertion. None of the polymerases tested are a universal bypass polymerase for the stereoisomeric bulky polycyclic aromatic hydrocarbon-DNA adducts derived from anti-BPDE.     

Structural basis of accurate replication beyond a bulky major benzo[a]pyrene adduct by human DNA polymerase kappa

Human Y-family DNA polymerase kappa (polκ) is specialized to bypass bulky lesions in DNA in an error-free way, thus protecting cells from carcinogenic bulky DNA adducts. Benzo[a]pyrene (BP) is one of the most ubiquitous polycyclic aromatic hydrocarbons and an environmental carcinogen. BP covalently modifies DNA and generates mutagenic, bulky adducts. The major BP adduct formed in cells is 10S (+)-trans-anti-BP-N²-dG adduct (BP-dG), which is associated with cancer. The molecular mechanism of how polκ replicates BP-dG accurately is not clear. Here we report the structure of polκ captured at the lesion-extension stage: the enzyme is extending the primer strand after the base pair containing the BP-dG adduct in the template strand at the −1 position. Polκ accommodates the BP adduct in the nascent DNA’s minor groove and keeps the adducted DNA helix in a B-form. Two water molecules cover the edge of the minor groove of the replicating base pair (0 position), which is secured by the BP ring in the −1 position in a 5′ orientation. The 5′ oriented BP adduct keeps correct Watson-Crick base pairing in the active site and promotes high fidelity replication. Our structural and biochemical data reveal a unique molecular basis for accurate DNA replication right after the bulky lesion BP-dG.     

Induction of specific base-pair substitutions in E. coli trpA mutants by chloroethylene oxide, a carcinogenic vinyl chloride metabolite

Chloroethylene oxide (CEO), an ultimate carcinogenic metabolite of vinyl chloride, induces base-pair substitution mutations but not frameshift mutations in bacteria. The mutational specificity of CEO was investigated in Escherichia coli, using the trpA mutants developed by Yanofsky. Reversion frequencies to tryptophan prototrophy were analysed, and CEO was found to induce more GC----AT transitions than AT----TA transversions, in addition to a low frequency of other types of substitution. This specificity indicates that CEO is mutagenic through a miscoding DNA adduct. The results are discussed in relation to the various CEO-DNA adducts formed and to their reported or expected mispairing properties.     

DNA binding and mutation spectra of the carcinogen N-2-aminofluorene in Escherichia coli. A correlation between the conformation of the premutagenic lesion and the mutation specificity

When the chemical carcinogen N-2-acetylaminofluorene binds to DNA in vivo, two major adducts are formed, both at position C-8 of the guanine residue. One of these (the acetylaminofluorene adduct) retains the acetyl group, while the other (the aminofluorene adduct) is the corresponding deacetylated form. Unlike -AAF adducts, which trigger important structural changes of the DNA secondary structure (either the insertion-denaturation model or the induction of a Z-DNA structure, depending upon the local nucleotide sequence), -AF adducts bind to the C-8 of guanine residues without causing any major conformational change of the B-DNA structure. Well-defined adducts (either -AF or -AAF) can be formed in vitro by reacting DNA with either N-hydroxy-N-2-aminofluorene or N-acetoxy-N-2-acetylaminofluorene. Specific cleavage of the phosphodiester backbone at -AF adducts can be achieved by treating -AF-modified DNA in 1 M-piperidine at 90 degrees C. This observation led us to construct the spectrum for -AF binding to a defined DNA restriction fragment. It is found that only guanine residues react to form alkali-labile lesions and that the reactivity among the different guanines is similar. In a forward mutation assay, namely the inactivation of the tetracycline resistance gene, we found previously that more than 90% of mutations induced by -AAF adducts are frameshift mutations. Using the same assay, we show here that -AF adducts induce primarily base substitution mutations (85%), mainly of the G to T transversion type. There is therefore a strong correlation between the nature of the carcinogen-induced conformational change of the DNA structure and the corresponding mutation specificity. The -AF-induced base substitution mutations depend upon the umuC gene function(s). The data obtained in our forward mutation assay are compared to the data previously obtained in the histidine reversion assay (Ames test).     

Mutational spectra for polycyclic aromatic hydrocarbons in the supF target gene

An SV40-based shuttle vector system was used to identify the types of mutational changes and the sites of mutation within the supF DNA sequence generated by the four stereoisomers of benzo[c]phenanthrene 3,4-dihydrodiol 1,2-epoxide (B[c]PhDE), by racemic mixtures of bay or fjord region dihydrodiol epoxides (DE) of 5-methylchrysene, of 5,6-dimethylchrysene, of benzo[g]chrysene and of 7-methylbenz[a]anthracene and by two direct acting polycyclic aromatic hydrocarbon carcinogens, 7-bromomethylbenz[a]anthracene (7-BrMeBA) and 7-bromomethyl-12-methylbenz[a]anthracene (7-BrMe-12-MeBA). The results of these studies demonstrated that the predominant type of mutation induced by these compounds is the base substitution. The chemical preference for reaction at deoxyadenosine (dAdo) or deoxyguanosine (dGuo) residues in DNA, which is in general correlated with the spatial structure (planar or non-planar) of the reactive polycyclic aromatic hydrocarbon, is reflected in the preference for mutation at AT or GC pairs. In addition, if the ability to react with DNA in vivo is taken into account, the relative mutagenic potencies of the B[c]PhDE stereoisomers are consistent with the higher tumorigenic activity associated with non-planar polycyclic aromatic hydrocarbons and their extensive reaction with dAdo residues in DNA. Comparison of the types of mutations generated by polycyclic aromatic hydrocarbons and other bulky carcinogens in this shuttle vector system suggests that all bulky lesions may be processed by a similar mechanism related to that involved in replication past apurinic sites. However, inspection of the distribution of mutations over the target gene induced by the different compounds demonstrated that individual polycyclic aromatic hydrocarbons induce unique patterns of mutational hotspots within the target gene. A polymerase arrest assay was used to determine the sequence specificity of the interaction of reactive polycyclic aromatic hydrocarbons with the shuttle vector DNA. The results of these assays revealed a divergence between mutational hotspots and polymerase arrest sites for all compounds investigated, i.e., sites of mutational hotspots do not correspond to sites where high levels of adduct formation occur, and suggested that some association between specific adducts and sequence context may be required to constitute a premutagenic lesion. A site-specific mutagenesis system employing a single-stranded vector (M13mp7L2) was used to investigate the mutational events a single benzo[a]pyrene or benzo[c]phenanthrene dihydrodiol epoxide–DNA adduct elicits within specific sequence contexts. These studies showed that sequence context can cause striking differences in mutagenic frequencies for given adducts. In addition, these sequence context effects do not originate only from nucleotides immediately adjacent to the adduct, but are also modulated by more distal nucleotides. The implications of these results for mechanisms of polycyclic aromatic hydrocarbon-induced mutagenesis and carcinogenesis are discussed.     

Ability of adducts formed in a shuttle vector by reactive metabolites of 1-nitropyrene and benzo(a)pyrene to induce mutations when the plasmid replicates in human cells

An SV40-based shuttle vector, pZ189, carrying a bacterial suppressor tRNA target gene (supF), was treated with radiolabeled polycyclic aromatic carcinogens and the number of covalently bound residues (adducts) per plasmid was determined. The plasmids were transfected into human cell line 293, allowed to replicate, and the progeny plasmids rescued and assayed for the frequency of supF mutants. The agents tested were the 7,8-diol-9,10-epoxide of benzo(a)pyrene (BPDE) and 1-nitrosopyrene (1-NOP). With each agent there was a linear increase in the frequency of supF mutants as a function of the number of DNA adducts formed, reaching frequencies 15 to 25 times higher than the background frequency of 1.4 x 10(-4). When compared on the basis of adducts formed per plasmid BPDE, which forms its principal DNA adduct at the N2 position of guanine, was approximately four times more mutagenic than 1-NOP, which binds principally at the C8 position of guanine. This difference in mutagenic effectiveness may reflect intrinsic differences in the nature of the adducts and their location in the DNA molecule, but it could also reflect a difference in the rate of removal of particular adducts by nucleotide excision repair since the 293 host cell line excised BPDE-induced adducts from genomic DNA at least three times slower than 1-NOP-induced adducts. Agarose gel electrophoresis and DNA sequencing analysis of mutants derived from untreated plasmids showed that the majority (70%) involved deletions, insertions, or altered gel mobility (gross rearrangements). In contrast, the majority of those derived from carcinogen-treated plasmids were base substitutions. DNA sequencing of 86 unequivocally independent mutants derived from BPDE-treated plasmid and 60 from 1-NOP-treated plasmid indicated that 70% to 80% contained a single base substitution, 5%-10% had two base substitutions, and 4%-10% had small insertions or deletions (one or two basepairs). The majority (83%) of the base substitutions in mutants from BPDE- or 1-NOP-treated plasmid were transversions, mainly G.C----T.A. Each carcinogen produced its own spectrum of mutations.     

Cytotoxicity and mutagenicity of aflatoxin dichloride in normal and repair deficient diploid human fibroblasts

The cytotoxic and mutagenic effect of aflatoxin B1-dichloride (AFB1-Cl2), a direct-acting carcinogen which is a model for the proposed ultimate reactive metabolite of AFB1 (the 2,3-epoxide), was compared in normal, repair-proficient, diploid human fibroblasts and in complementation Group A xeroderma pigmentosum cells (XP12BE) which are virtually incapable of excision repair of DNA damage induced by ultraviolet radiation, the 7,8-diol-9,10-epoxide of benzo[alpha]pyrene, and several reactive aromatic amide derivatives. The XP cells were significantly more sensitive than normal to the cytotoxic and mutagenic effects of AFB1-Cl2, not only as a function of concentration administered but also of the number of AFB1-Cl2 residues initially bound to DNA. Cytotoxicity was determined from survival of colony-forming ability; resistance to 6-thioguanine was the genetic marker used for mutagenicity. We compared the rate of loss of AFB1-Cl2-DNA adducts from cells treated and held in the non-dividing state (confluent) over several days, as well as their ability to recover from the potentially mutagenic and/or cytotoxic effects of the agent. AFB1-Cl2 residues were lost from both strains of cells and both exhibited a gradual increase in survival. However, the rate of loss of adducts from the DNA in the normal cells was more rapid than in XP cells and they exhibited recovery from higher doses of AFB1-Cl2 than XP cells. The major primary DNA adduct formed in the human cells and in isolated DNA was a chemically unstable guanine derivative which could undergo a change in structure with time posttreatment to form a more stable secondary adduct. The cytotoxic effect of AFB1-Cl2 was highly correlated with the presence of either of these guanine adducts. Evidence suggests that the primary adduct is an N7-guanine adduct. The kinetics of the loss of this guanine and its transformation into the more stable secondary adduct resembled that reported recently for the major primary DNA adduct formed by the reaction of AFB1 at the N-7 position of guanine in the DNA of normal and XP cells and its transformation into the putative AFB1-ring opened triamino pyrimidyl structure.     

Adducts formed by the food mutagen 2-amino-3-methylimidazo(4, 5-f ) quinoline induce frameshift mutations at hot spots through an SOS-independent pathway

The potency of 2-amino-3-methylimidazo(4, 5-f)quinoline (IQ) adducts to induce ?2, ?1 and +1 frameshift mutations has been determined on specific target DNA sequences, namely short runs of alternating GpC sequences and short runs of guanines. The genetic control of the mutational processes has been analyzed using different Escherichia coli mutants, affected either in the control or in the mutagenesis pathway of the SOS system. We have shown that IQ adducts induce very efficiently both ?1 and ?2 frameshift mutations in E. coli. Both types of deletion mutations are induced in bacteria without the need of SOS induction, indicating that no LexA-controlled functions, in particular the UmuDC proteins, are required for mutation fixation. We have also shown that the frequency of IQ-induced ?2 frameshift mutations in alternating GC sequences increases with the length of the repetition. The efficiency of IQ adducts to induce ?1 and ?2 frameshift mutations is similar to that of N‐2-acetylaminofluorene (AAF) adducts. Both chemicals are potent carcinogens which form covalent adducts at the C8 position of guanines. We suggest that in both cases the adduct-induced DNA structure allows the replication complex to perform a mutagenic bypass of the lesion by a slippage mechanism. However, in contrast to AAF-induced frameshift mutagenesis, IQ-induced frameshift mutagenesis is SOS-independent.     

Mutational spectrum induced in Saccharomyces cerevisiae by the carcinogen N-2-acetylaminofluorene

The spectrum of mutations induced by the carcinogen N-2-acetylaminofluorene (AAF) was analysed in Saccharomyces cerevisiae using a forward mutation assay, namely the inactivation of the URA3 gene. The URA3 gene, carried on a yeast/bacterial shuttle vector, was randomly modified in vitro using N-acetoxy-N-2-acetylaminofluorene (N-AcO-AAF) as a model reactive metabolite of the carcinogen AAF. The binding spectrum of AAF to the URA3 gene was determined and found to be essentially random, as all guanine residues reacted about equally well with N-AcO-AAF. Independent Ura^? mutants were selected in vivo after transformation of the modified plasmid into a ura3Δ yeast strain. Plasmid survival decreased as a function of AAF modification, leading to one lethal hit (37% relative survival) for an average of ≈ 50 AAF adducts per plasmid molecule. At this level of modification the mutation frequency was equal to ≈ 70 × 10^?4, i.e. ≈ 50-fold above the background mutation frequency. UV irradiation of the yeast cells did not further stimulate the mutagenic response, indicating the lack of an SOS-like mutagenic response in yeast. Sequence analysis of the URA3 mutants revealed ≈ 48% frameshifts, 44% base substitutions and ≈ 8 % complex events. While most base substitutions (74%) were found to be targeted at G residues where AAF is known to form covalent C8 adducts, frameshift mutations were observed at GC base pairs in only≈ 24% of cases. Indeed, more than 60% of frameshift events occurred at sequences such as 5′-(A/T)_nG-3′ where a short (n = 2 or 3) monotonous run of As or Ts is located on the 5' side of a guanine residue. We refer to these mutations as semi-targeted events and present a potential mechanism that explains their occurrence.     

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.     

Complex Frameshift Mutations Mediated by Plasmid Pkm101: Mutational Mechanisms Deduced from 4-Aminobiphenyl-Induced Mutation Spectra in Salmonella

We used colony probe hybridization and polymerase chain reaction/DNA sequence analysis to determine the mutations in ~2,400 4-aminobiphenyl (4-AB) +S9-induced revertants of the -1 frameshift allele hisD3052 and of the base-substitution allele hisG46 of Salmonella typhimurium. Most of the mutations occurred at sites containing guanine, which is the primary base at which 4-AB forms DNA adducts. A hotspot mutation involving the deletion of a CG or GC within the sequence CGCGCGCG accounted for 100 and 99.9%, respectively, of the reversion events at the hisD3052 allele in the pKM101 plasmid-minus strains TA1978 (uvr(+)) and TA1538 (δuvrB). In strain TA98 (δuvrB, pKM101), which contained the SOS DNA repair system provided by the pKM101 plasmid, ~85% of the revertants also contained the hotspot deletion; the remaining ~15% contained one of two types of mutations: (1) complex frameshifts that can be described as a -2 or + 1 frameshift and an associated base substitution and (2) deletions of the CC or GG sequences that flank the hotspot site (CCGCGCGCGG). We propose a misincorporation/slippage model to account for these mutations in which (1) pKM101-mediated misincorporation and translesion synthesis occurs across a 4-AB-adducted guanine; (2) the instability of such a mispairing and/or the presence of the adduct leads to strand slippage in a run of repeated bases adjacent to the adducted guanine; and (3) continued DNA synthesis from the slipped intermediate produces a frameshift associated with a base substitution. This model readily accounts for the deletion of the CC or GG sequences flanking the hotspot site, indicating that these mutations are, in fact, complex mutations in disguise (i.e., cryptic complex frameshifts). The inferred base-substitution specificity associated with the complex frameshifts at the hisD3052 allele (primarily G·C -> T·A transversions) is consistent with the finding that 4-AB induced primarily G·C -> T·A transversions at the hisG46 base-substitution allele. The model also provides a framework for understanding the different relative mutagenic potencies of 4-AB at the two alleles in the various DNA repair backgrounds of Salmonella.     

Structure and stability of DNA containing an aristolactam II-dA lesion: implications for the NER recognition of bulky adducts

Aristolochic acids I and II are prevalent plant toxicants found in the Aristolochiaceae plant family. Metabolic activation of the aristolochic acids leads to the formation of a cyclic N-hydroxylactam product that can react with the peripheral amino group of purine bases generating bulky DNA adducts. These lesions are mutagenic and established human carcinogens. Interestingly, although AL-dG adducts progressively disappear from the DNA of laboratory animals, AL-dA lesions has lasting persistence in the genome. We describe here NMR structural studies of an undecameric duplex damaged at its center by the presence of an ALII-dA adduct. Our data establish a locally perturbed double helical structure that accommodates the bulky adduct by displacing the counter residue into the major groove and stacking the ALII moiety between flanking bases. The presence of the ALII-dA perturbs the conformation of the 5'-side flanking base pair, but all other pairs of the duplex adopt standard conformations. Thermodynamic studies reveal that the lesion slightly decreases the energy of duplex formation in a sequence-dependent manner. We discuss our results in terms of its implications for the repair of ALII-dA adducts in mammalian cells.     

Kinds and spectrum of mutations induced by 1-nitrosopyrene adducts during plasmid replication in human cells.

1-Nitropyrene has been shown in bacterial assays to be the principal mutagenic agent in diesel emission particulates. It has also been shown to be mutagenic in human fibroblasts and carcinogenic in animals. To investigate the kinds of mutations induced by this carcinogen and compare them with those induced by a structurally related carcinogen, (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetra-hydrobenzo [a]pyrene (BPDE) (J.-L. Yang, V. M. Maher, and J. J. McCormick, Proc. Natl. Acad. Sci. USA 84:3787-3791, 1987), we treated a shuttle vector with tritiated 1-nitrosopyrene (1-NOP), a carcinogenic mutagenic intermediate metabolite of 1-nitropyrene which forms the same DNA adduct as the parent compound, and introduced the plasmids into a human embryonic kidney cell line, 293, for DNA replication to take place. The treated plasmid, pZ189, carrying a bacterial suppressor tRNA target gene, supF, was allowed 48 h to replicate in the human cells. Progeny plasmids were then rescued, purified, and introduced into bacteria carrying an amber mutation in the beta-galactosidase gene in order to detect those carrying mutations in the supF gene. The frequency of mutants increased in direct proportion to the number of DNA-1-NOP adducts formed per plasmid. At the highest level of adduct formation tested, the frequency of supF mutants was 26 times higher than the background frequency of 1.4 X 10(-4). DNA sequencing of 60 unequivocally independent mutant derived from 1-NOP-treated plasmids indicated that 80% contained a single base substitution, 5% had two base substitutions, 4% had small insertions or deletions (1 or 2 base pairs), and 11% showed a deletion or insertion of 4 or more base pairs. Sequence data from 25 supF mutants derived from untreated plasmids showed that 64% contained deletions of 4 or more base pairs. The majority (83%) of the base substitution in mutants from 1-NOP-treated plasmids were transversions, with 73% of these being G . C --> T . A. This is very similar to what we found previously in this system, using BPDE, but each carcinogen produced its own spectrum of mutations. Of the five hot spots for base substitution mutations produced in the supF gene with 1-NOP, two were the same as seen with BPDE-treated plasmids. However, the three other hot spots were cold spots for BPDE-treated plasmids. Conversely, four of the other five hot spots seen with BPDE-treated plasmids were cold spots for 1-NOP-treated plasmids. Comparison of the two carcinogens for the frequency of supF mutants induced per DNA adduct showed that 1-NOP-induced adducts were 3.8 times less than BPDE adducts. However, the 293 cell excised 1-NOP-induced adducts faster than BPDE adducts.     

The 4-nitroquinoline 1-oxide mutational spectrum in single stranded DNA is characterized by guanine to pyrimidine transversions.

4-Nitroquinoline-1-oxide is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. In ds or ss damaged DNA the ratio C8/N2 adducts is 1:2 and 8-10:1, respectively. In bacteria and yeast 4NQO has been shown to be a base substitution mutagen acting at G residues inducing mainly G to A transitions. We determined the mutational spectrum induced by the 4NQO metabolite, acetoxy-4-aminoquinoline 1-oxide, in the M13lacZ'/E. coli lacZ delta M15 alpha complementation assay using ssDNA. Among 68 Ac-4HAQO induced mutants, G to Pyr transversion was the most frequent base substitution observed. By comparison with dsDNA based systems, our data suggest that dGuo-C8-AQO induces G to Pyr transversions. A mechanism to explain how this lesion may induce transversions is proposed.     

Site-specific modification of the lactose operator with acetylaminofluorene

We have synthesized the tetradecamer GAGCXGATAACAAG containing a part of the sequence of the lactose operator. A guanine base in the sequence is replaced by the adduct of the carcinogen 2-acetylaminofluorene with guanine. Under the standard conditions of de-protection, the fluorene moiety is lost, leaving behind a guanine oxidation product. New conditions of de-protection have been developed which allow the isolation of an oligonucleotide containing the adduct of 2-aminofluorene with guanine. The presence of the aminofluorene adduct greatly increases retention on reverse phase chromatography and produces a unique pattern of sequencing bands.     

Extending the understanding of mutagenicity: structural insights into primer-extension past a benzo[a]pyrene diol epoxide-DNA adduct

DNA polymerase enzymes employ a number of innate fidelity mechanisms to ensure the faithful replication of the genome. However, when confronted with DNA damage, their fidelity mechanisms can be evaded, resulting in a mutation that may contribute to the carcinogenic process. The environmental carcinogen benzo[a]pyrene is metabolically activated to reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide, which can attack DNA at the exocyclic amino group of guanine to form the major (+)-trans-anti-[BP]-N(2)-dG adduct. Bulky adducts such as (+)-trans-anti-[BP]-N(2)-dG primarily block DNA replication, but are occasionally bypassed and cause mutations if paired with an incorrect base. In vitro standing-start primer-extension assays show that the preferential insertion of A opposite (+)-trans-anti-[BP]-N(2)-dG is independent of the sequence context, but the primer is extended preferentially when dT is positioned opposite the damaged base in a 5'-CG*T-3' sequence context. Regardless of the base positioned opposite (+)-trans-anti-[BP]-N(2)-dG, extension of the primer past the lesion site poses the greatest block to polymerase progression. In order to gain insight into primer-extension of each base opposite (+)-trans-anti-[BP]-N(2)-dG, we carried out molecular modeling and 1.25 ns unrestrained molecular dynamics simulations of the adduct in the +1 position of the template within the replicative pol I family T7 DNA polymerase. Each of the four bases was modeled at the 3' terminus of the primer, incorporated opposite the adduct, and the next-to-be replicated base was in the active site with its Watson-Crick partner as the incoming nucleotide. As in our studies of nucleotide incorporation, (+)-trans-anti-[BP]-N(2)-dG was modeled in the syn conformation in the +1 position, with the BP moiety on the open major groove side of the primer-template duplex region, leaving critical protein-DNA interactions intact. The present work revealed that the efficiency of primer-extension past this bulky adduct opposite each of the four bases in the 5'-CG*T-3' sequence can be rationalized by the stability of interactions between the polymerase protein, primer-template DNA and incoming nucleotide. However, the relative stabilization of each nucleotide opposite (+)-trans-anti-[BP]-N(2)-dG in the +1 position (T > G > A > or = C) differed from that when the adduct and partner were the nascent base-pair (A > T > or = G > C). In addition, extension past (+)-trans-anti-[BP]-N(2)-dG may pose a greater block to a high fidelity DNA polymerase than does nucleotide incorporation opposite the adduct because the presence of the modified base-pair in the +1 position is more disruptive to the polymerase-DNA interactions than it is within the active site itself. The dN:(+)-trans-anti-[BP]-N(2)-dG base-pair is strained to shield the bulky aromatic BP moiety from contact with the solvent in the +1 position, causing disruption of protein-DNA interactions that would likely result in decreased extension of the base-pair. These studies reveal in molecular detail the kinds of specific structural interactions that determine the function of a processive DNA polymerase when challenged by a bulky DNA adduct.