The precautionary principle in environmental science.

Environmental scientists play a key role in society's responses to environmental problems, and many of the studies they perform are intended ultimately to affect policy. The precautionary principle, proposed as a new guideline in environmental decision making, has four central components: taking preventive action in the face of uncertainty; shifting the burden of proof to the proponents of an activity; exploring a wide range of alternatives to possibly harmful actions; and increasing public participation in decision making. In this paper we examine the implications of the precautionary principle for environmental scientists, whose work often involves studying highly complex, poorly understood systems, while at the same time facing conflicting pressures from those who seek to balance economic growth and environmental protection. In this complicated and contested terrain, it is useful to examine the methodologies of science and to consider ways that, without compromising integrity and objectivity, research can be more or less helpful to those who would act with precaution. We argue that a shift to more precautionary policies creates opportunities and challenges for scientists to think differently about the ways they conduct studies and communicate results. There is a complicated feedback relation between the discoveries of science and the setting of policy. While maintaining their objectivity and focus on understanding the world, environmental scientists should be aware of the policy uses of their work and of their social responsibility to do science that protects human health and the environment. The precautionary principle highlights this tight, challenging linkage between science and policy.

Factors that influence the mutagenic patterns of DNA adducts from chemical carcinogens.

Carcinogens are generally mutagens, which is understandable given that tumor cells grow uncontrollably because they have mutations in critical genes involved in growth control. Carcinogens often induce a complex pattern of mutations (e.g., GC-->TA, GC-->AT, etc.). These mutations are thought to be initiated when a DNA polymerase encounters a carcinogen-DNA adduct during replication. In principle, mutational complexity could be due to either a collection of different adducts each inducing a single kind of mutation (Hypothesis 1a), or a single adduct inducing different kinds of mutations (Hypothesis 1b). Examples of each are discussed. Regarding Hypothesis 1b, structural factors (e.g., DNA sequence context) and biological factors (e.g., differing DNA polymerases) that can affect the pattern of adduct mutagenesis are discussed. This raises the question: how do structural and biological factors influence the pattern of adduct mutagenesis. For structural factors, three possibilities are considered: (Hypothesis 2a) a single conformation of an adduct giving rise to multiple mutations -- dNTP insertion by DNA polymerase being influenced by (e.g.) the surrounding DNA sequence context; (Hypothesis 2b) a variation on this ("dislocation mutagenesis"); or (Hypothesis 2c) a single adduct adopting multiple conformations, each capable of giving a different pattern of mutations. Hypotheses 2a, 2b and 2c can each in principle rationalize many mutational results, including how the pattern of adduct mutagenesis might be influenced by factors, such as DNA sequence context. Five lines of evidence are discussed suggesting that Hypothesis 2c can be correct for base substitution mutagenesis. For example, previous work from our laboratory was interpreted to indicate that [+ta]-B[a]P-N(2)-dG in a 5'-CGG sequence context (G115) could be trapped in a conformation giving predominantly G-->T mutations, but heating caused the adduct to equilibrate to its thermodynamic mixture of conformations, leading to a decrease in the fraction of G-->T mutations. New work is described suggesting that [+ta]-B[a]P-N(2)-dG at G115 can also be trapped predominantly in the G-->A mutational conformation, from which equilibration can also occur, leading to an increase in the fraction of G-->T mutations. Evidence is also presented that the fraction of G-->T mutations is higher when [+ta]-B[a]P-N(2)-dG at G115 is in ss-DNA ( approximately 89%) vs. ds-DNA ( approximately 66%), a finding that can be rationalized if the mixture of adduct conformations is different in ss- and ds-DNA. In summary, the factors affecting adduct mutagenesis are reviewed and five lines of evidence that support one hypothesis (2c: adduct conformational complexity can cause adduct mutational complexity) are discussed.

Toward an understanding of the role of DNA adduct conformation in defining mutagenic mechanism based on studies of the major adduct (formed at N(2)-dG) of the potent environmental carcinogen, benzo[a]pyrene.

The process of carcinogenesis is initiated by mutagenesis, which often involves replication past damaged DNA. One question - what exactly is a DNA polymerase seeing when it incorrectly copies a damaged DNA base (e.g., inserting dATP opposite a dG adduct)? - has not been answered in any case. Herein, we reflect on this question, principally by considering the mutagenicity of one activated form of benzo[a]pyrene, (+)-anti-B[a]PDE, and its major adduct [+ta]-B[a]P-N(2)-dG. In previous work, [+ta]-B[a]P-N(2)-dG was shown to be capable of inducing>95% G-->T mutations in one sequence context (5'-TGC), and approximately 95% G-->A mutations in another (5'-AGA). This raises the question - how can a single chemical entity induce different mutations depending upon DNA sequence context? Our current working hypothesis is that adduct conformational complexity causes adduct mutational complexity, where DNA sequence context can affect the former, thereby influencing the latter. Evidence supporting this hypothesis was discussed recently (Seo et al., Mutation Res. [in press]). Assuming this hypothesis is correct (at least in some cases), one goal is to consider what these mutagenic conformations might be. Based on molecular modeling studies, 16 possible conformations for [+ta]-B[a]P-N(2)-dG are proposed. A correlation between molecular modeling and mutagenesis work suggests a hypothesis (Hypothesis 3): a base displaced conformation with the dG moiety of the adduct in the major vs. minor groove gives G-->T vs. G-->A mutations, respectively. (Hypothesis 4, which is a generalized version of Hypothesis 3, is also proposed, and can potentially rationalize aspects of both [+ta]-B[a]P-N(2)-dG and AP-site mutagenesis, as well as the so-called "A-rule".) Finally, there is a discussion of how conformational complexity might explain some unusual mutagenesis results that suggest [+ta]-B[a]P-N(2)-dG can become trapped in different conformations, and why we think it makes sense to interpret adduct mutagenesis results by modeling ds-DNA (at least in some cases), even though the mutagenic event must occur at a ss/ds-DNA junction in the presence of a DNA polymerase.

DNA guanine-guanine crosslinking sequence specificity of isophosphoramide mustard, the alkylating metabolite of the clinical antitumor agent ifosfamide.

PURPOSE: The purpose of this investigation was to determine the base sequence specificity of isophosphoramide mustard (IPM), the alkylating metabolite of ifosfamide, by crosslinking of designed DNA oligomers in comparison with the clinical alkylating agents mechlorethamine (ME) (nitrogen mustard) and phosphoramide mustard (PM), the alkylating metabolite of cyclophosphamide. METHODS: IPM, as well as PM and ME were each reacted with three dodecameric duplexes, which were designed to detect interstrand crosslinking between guanines in 5'-GC-3' (I), 5'-GNC-3' (II) or 5'-GNNC-3' (III) sequences (N = A or T). RESULTS: All three agents preferentially react with 5'-GNC-3' target sequences. The 5'-GNNC-3' target sequence is less reactive by a factor of approximately 2.5- to 10-fold, while 5'-GC-3' is of even lower reactivity. CONCLUSION: These results indicate that all three agents show approximately equal preference for reaction with a 5'-GNC-3' target sequence in spite of the fact that IPM yields a 7-atom crosslink, while the other two agents yield 5-atom crosslinks.

DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli.

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0. 6) than in a DeltapolB (approximately 0.1) strain. Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.

The major, N2-dG adduct of (+)-anti-B[a]PDE induces G-->A mutations in a 5'-AGA-3' sequence context.

Previously, in a random mutagenesis study, the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] was shown to induce a complex mutational spectrum in the supF gene of an Escherichia coli plasmid, which included insertions, deletions and base substitution mutations, notably a significant fraction of GC-->TA, GC-->AT and GC-->CG mutations. At some sites, a single type of mutation dominated and to understand individual mutagenic pathways these sites were chosen for study by site-specific means to determine whether the major adduct, [+ta]-B[a]P-N2-dG, was responsible. [+ta]-B[a]P-N2-dG was shown to induce approximately 95% G-->T mutations in a 5'-TGC-3' sequence context and approximately 80% G-->A mutations in a 5'-CGT-3' sequence context. (+)-anti-B[a]PDE induced principally GC-->CG mutations in the G133 sequence context (5'-AGA-3') in studies using both SOS-uninduced or SOS-induced E. coli. Herein, [+ta]-B[a]P-N2-dG is shown to induce principally G-->A mutations (>90%) either without or with SOS induction in a closely related 5'-AGA-3' sequence context (identical over 7 bp). This is the first time that there has been a discrepancy between the mutagenic specificity of (+)-anti-B[a]PDE versus [+ta]-B[a]P-N2-dG. Eight explanations for this discordance are considered. Four are ruled out; e.g. the second most prevalent adduct [+ca]-B[a]P-N2-dG also induces a preponderance of G-->A mutations (>90%), so it also is not responsible for (+)-anti-B[a]PDE-induced G133-->C mutations. The four explanations not ruled out are discussed and include that another minor adduct might be responsible and that the 5'-AGA-3' sequence context differed slightly in the studies with [+ta]-B[a]P-N2-dG versus (+)-anti-B[a]PDE. In spite of the discordance, [+ta]-B[a]P-N2-dG induces G-->A mutations in the context studied herein and this result has proven useful in generating a hypothesis for what conformations of [+ta]-B[a]P-N2-dG are responsible for G-->T versus G-->A mutations.

Molecular modeling of the major adduct of (+)-anti-B[a]PDE (N2-dG) in the eight conformations and the five DNA sequences most relevant to base substitution mutagenesis.

The potent mutagen/carcinogen 7R,8S-dihydroxy-9S, 10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-B[a]PDE], which is the activated form of benzo[a]pyrene (B[a]P), is able to induce different kinds of mutations (G-->T, G-->A, etc.). One hypothesis for this is that different mutations are induced depending upon the conformation of its major adduct ([+ta]-B[a]P-N2-dG) when bypassed during DNA replication. Based on molecular modeling, there appear to be at least 16 potential conformations that the major adduct [+ta]-B[a]P-N2-dG can adopt in dsDNA. Regarding base substitution mutagenesis, eight conformations are most likely to be relevant. In two conformations the dG moiety of the adduct is base paired with its complementary dC and the B[a]P moiety is in the minor groove. In two others the dG moiety of the adduct is in the Hoogsteen orientation and the B[a]P moiety is in the major groove. There are four base displaced structures in which the B[a]P moiety of the adduct is stacked with the surrounding base pairs, two with dG in the major groove and two with dG in the minor groove. Using a simulated annealing protocol, these eight conformations were evaluated in five different DNA sequence contexts (5'-TGC-3', 5'-CGT-3', 5'-AGA-3', 5'-CGG-3' and 5'-GGG-3'); the latter were chosen because they may be particularly revealing about mutagenic mechanism based on studies with [+ta]-B[a]P-N2-dG and (+)-anti-B[a]PDE. For each conformation and each sequence context, 25 simulated annealing runs were conducted by systematically varying several parameters (such as the initial annealing temperature) based on a protocol established recently. The goal of this work was to exclude conformations that are clearly inferior. Three conformations are virtually always high in energy, including the two Hoogsteen oriented species and one of the base displaced species with dG in the major groove. Remarkably, the remaining five conformations are often quite close in energy and are deemed most likely to be relevant to mutagenesis (see accompanying paper).

A hypothesis for what conformation of the major adduct of (+)-anti-B[a]PDE (N2-dG) causes G-->T versus G-->A mutations based upon a correlation between mutagenesis and molecular modeling results.

Molecular modeling (simulated annealing) was used to study the conformations in dsDNA of [+ta]-B[a]P-N2-dG (R.E. Kozack and E.L.Loechler, accompanying paper), which is the major benzo[a]pyrene (B[a]P) adduct. Sixteen classes of conformations were identified, and are analyzed herein vis-a-vis the two most prominent B[a]P mutations, G-->T and G-->A base substitutions. Eight conformations seem more relevant to frameshift mutagenesis, so they are excluded, leaving eight conformations as follows. Two conformations (BPmi5 and BPmi3) retain Watson-Crick G:C base pairing having the B[a]P moiety of the adduct in the minor groove. Two conformations (BPma5 and BPma3) have the Hoogsteen orientation with B[a]P in the major groove. Four conformations are base displaced and have B[a]P stacked in the helix with the dG moiety of the adduct displaced into either the major groove (Gma5 and Gma3) or the minor groove (Gmi5 and Gmi3). Three of these eight conformations (BPma5, BPma3 and Gma3) are universally high in energy. The two conformations that retain G:C base pairing potential (BPmi5 and BPmi3) are likely to be non-mutagenic. Of the three remaining conformations, Gmi5 can be relatively low in energy, but is distorted. A correlation exists between the calculated energies for the remaining two base displaced conformations and mutagenesis for [+ta]-B[a]P-N2-dG, leading to the hypothesis that Gma5 is responsible for G-->T mutations and Gmi3 is responsible for G-->A mutations. Gma5 and Gmi3 resemble each other, except that dG is in the major and minor grooves, respectively. An incipient rationale for this hypothesis is discussed: DNA polymerase might be triggered to follow a different mutagenic pathway depending upon whether a non-informational lesion has bulk protruding into the major or minor groove. A pathway for interconversion between these eight conformations is also proposed and its implications are discussed; e.g. four steps are required to interconvert between Gma5 and Gmi3.

How stereochemistry affects mutagenesis by N2-deoxyguanosine adducts of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene: configuration of the adduct bond is more important than those of the hydroxyl groups.

Previous work has shown that the major adduct from the (+)-anti diol epoxide of benzo[a]pyrene (B[a]P), which forms at N2-deoxyguanosine [(+)-trans-anti-B[a]P-N2-dG], is capable of inducing either predominantely G --> T mutations ( approximately 95%) in a 5'-TGC-3 sequence context or predominantly G --> A mutations ( approximately 80%) in a 5'-CGT-3' sequence context. This is likely to be attributable to the major adduct being in a different mutagenic conformation in each case. In the next phase of this work, the questions to be addressed are what conformation is associated with what mutation and why? To help define what aspect of adduct structure is important to mutagenesis, the work herein reports on the mutations induced in a single sequence context by four stereoisomers of B[a]P-N2-dG: (+)-trans-, (+)-cis-, (-)-trans-, and (-)-cis-. The (+)-trans- and (-)-cis-adducts show a remarkably similar mutational pattern with G --> A mutations predominating ( approximately 80%). The (-)-trans- and (+)-cis-adducts also show a similar mutational pattern with a more even mixture of G --> T, G --> A, and G --> C mutations. Each of these adducts has an adduct bond and three hydroxyl groups at four consecutive saturated carbons in the B[a]P moiety of the adduct; the stereochemistry at these four positions differs in each of the adducts. The (+)-trans- and (-)-cis-adducts are a pair sharing the S configuration for the adduct bond, although they are a mirror image vis-a-vis the hydroxyl groups. The (-)-trans- and (+)-cis-adducts share the opposite adduct bond stereochemistry (R) but differ in the stereochemistry of their hydroxyl groups. Thus, there is a correlation suggesting that anti-B[a]P-N2-dG adduct mutagenesis is more dependent on the stereochemistry of the adduct bond than on the stereochemistry of the hydroxyl groups.

The major, N2-dG adduct of (+)-anti-B[a]PDE shows a dramatically different mutagenic specificity (predominantly, G --> A) in a 5'-CGT-3' sequence context.

Mutations induced by the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] were described previously in the supF gene of the Escherichia coli plasmid pUB3 [Rodriguez et al.(1993) Biochemistry, 32, 1759]. (+)-anti-B[a]PDE induced a complex pattern of mutations, including insertions, deletions, frameshifts, as well as base substitution mutations, which for G:C base pairs alone included a significant fraction of G:C --> T:A, A:T and C:G mutations. A variety of results suggest that most of these mutations arise from the major adduct ([+ta]-B[a]P-N2-dG), raising the question how can a single adduct induce different kinds of mutations? Our working hypothesis in this regard is that (1) an adduct can adopt multiple conformations; (2) different conformations cause different mutations; and (3) adduct conformation is controlled by various factors, such as DNA sequence context. To investigate what conformation is associated with what mutation, it is essential to find examples where [+ta]-B[a]P-N2-dG induces principally one kind of mutation as a prelude to the study in that same context of the conformation(s) potentially relevant to mutagenesis. Earlier work indicated that (+)-anti-B[a]PDE gave a preponderance of G --> A mutations in a 5'-CGT-3 sequence context, and herein it is shown that these mutations are likely to be attributable to the major adduct, since in this same sequence context [+ta]-B[a]P-N2-dG studied site specifically also induces principally G --> A mutations ( approximately 82%). Previously, [+ta]-B[a]P-N2-dG was shown to induce principally G --> T mutations (approximately 97%) in a 5'-TGC-3' sequence context. Thus, by simply altering its surrounding sequence context this adduct can give a preponderance of either G --> A or G --> T mutations. This is the most dramatic change in base substitution mutagenic specificity for an adduct described to date and illustrates that the qualitative pattern of mutagenesis by a bulky adduct can be remarkably diverse.

Molecular modeling of the conformational complexity of (+)-anti-B[a]PDE-adducted DNA using simulated annealing.

Benzo[a]pyrene (B[a]P), a potent mutagen/carcinogen, reacts with DNA following metabolism to its corresponding (+)-anti-7,8-diol-9,10-epoxide [(+)-anti-B[a]PDE], giving a major adduct (+)-trans-anti-B[a]P-N2-dG. Evidence suggests that this adduct is responsible for most of the different kinds of mutations (e.g. G-->T, G-->A, etc.) induced by (+)-anti-B[a]PDE, raising the question of how can a single adduct cause many different kinds of mutations? One hypothesis is that different mutations are induced depending upon the conformation of this adduct when bypassed during DNA replication. If true, then it becomes imperative to explore different reasonable conformations for this adduct. Herein a simulated annealing protocol is employed to study the conformation of (+)-trans-anti-B[a]P-N2-dG with the B[a]P moiety in the minor groove and pointing toward the base on its 5'-side in a 5'-CGC-3' sequence context in duplex DNA. This conformation and sequence were chosen because there is a structure derived from NMR constraints for comparison. A four step procedure is followed: the adduct is docked in canonical B-DNA, after which the structure is subjected to an initial conjugate gradient minimization, followed by simulated annealing and a final conjugate gradient minimization. The quality and final energy of structures is assessed as a function of changes in six parameters, including the length of the DNA helix, the initial annealing temperature (T0), the annealing time (t), the molecular dynamics time step (tau) and two other parameters. While there is no single set of optimum parameters, reasonable low energy structures were obtained using the values t approximately 40 ps (or longer), T0 approximately 750 K and tau approximately 1.0 fs with a helix length of 7 bp. The structures that emerge all retain the basic features of the input structure, being B-DNA-like with the B[a]P moiety in the minor groove pointing toward the base on the 5'-side. However, within this broad category there are at least six subclasses of structures, of which four have lowest energy members that differ by < approximately 5 kcal/mol. The fact that a variety of distinct but related structures emerge from a single starting structure as this parameter set is varied suggests that the use of a large but manageable number of simulated annealing runs should be considered in the search for a cohort of related structures. This is especially important given that this breadth of potentially relevant structures of approximately the same energy may indeed be relevant to the hypothesis that different mutations arise from a single adduct in different conformations.

Sequence specific mutagenesis of the major (+)-anti-benzo[a]pyrene diol epoxide-DNA adduct at a mutational hot spot in vitro and in Escherichia coli cells.

In the supF gene, most (+)-anti-benzo[a]pyrene diol epoxide ((+)-anti-B[a]PDE) mutagenesis hot spots in Escherichia coli are in 5'-GG sequences [Rodriguez and Loechler (1993) Carcinogenesis 14, 373-383]. A major hot spot was detected at G1 in the sequence 5'-GCG1G2-CCAAAG, whereas G2 yielded very few mutants. In order to investigate the details of such sequence context effects of (+)-anti-B[a]PDE mutagenesis, we have constructed 25-mer oligonucleotides and single-stranded M13 genomes containing the above decamer sequence, in which the trans-N2-dG adduct induced by (+)-anti-B[a]PDE [(+)-trans-anti-B[a]P-N2-dG] at G1 or G2 was introduced. In vitro DNA synthesis on the adducted 25-mers was strongly blocked at each site, although the 3'-->5' exonuclease-deficient Klenow fragment could incorporate a nucleotide opposite the adduct in the presence of Mn2+. For both sites purine nucleotides were preferred. The ratio Vmax/K(m) indicated that the efficiency of incorporation of dGTP opposite these sites was very similar, but dATP incorporation opposite the adduct at G1 was five-fold more efficient than that at G2. For each site, further extension beyond the adducted nucleotide was investigated by annealing four different primers, in which only the nucleotide opposite the adducted deoxyguanosine was altered. Significant extension was only observed when deoxyadenosine was located opposite adducted G1. When the M13 genomes containing the (+)-trans-anti-B[a]P-N2-dG were replicated in E. coli, survival of each adducted genome was less than 1% as compared to the unadducted genome. Upon induction of SOS, viability increased 2-6-fold. DNA sequencing showed no base substitutions in the progeny from SOS-uninduced cells, although small deletions in a quasipalindromic sequence occurred with the adduct being located at either site. However, following SOS induction, up to 40% targeted base substitutions were detected when the adduct was located at G1, while approximately 12% of the progeny were mutants with the adduct at G2. Most base substitutions were targeted G-->T transversions. We conclude that (+)-trans-anti-B[a]P-N2-dG is a highly mutagenic and replication blocking lesion. In addition, the biological consequence of this adduct depends on whether it is located at G1 or G2, suggesting that sequence context plays a major role in the mutagenic processing of this adduct.

Evidence for a recombination-independent pathway for the repair of DNA interstrand cross-links based on a site-specific study with nitrogen mustard.

DNA-DNA interstrand cross-links are thought to be important for the cytotoxicity of many chemotherapeutic agents. To study this more definitively, adduct site-specific methods are used to construct a plasmid with a single nitrogen mustard interstrand cross-link (inter-HN2-pTZSV28). Replication efficiency (RE = [colonies from (inter-HN2-pTZSV28)/(control with no cross-link)]) is approximately 0.3 following transformation into Escherichia coli, implying that the cross-link is repaired. The commonly accepted pathway for cross-link repair, which involves both nucleotide excision repair (NER) and recombination, is ruled out since RE is approximately 0.3 in a delta recA strain. Non-RecA-directed recombination such as copy-choice is also unlikely. However, NER is involved since RE was approximately 0.02 in strains deficient in NER. Base excision repair is not important since RE is approximately 0.3 in strains deficient in 3-methyladenine DNA glycosylases I and II, FAPY DNA glycosylase, both known apurinic/apyrimidinic endonucleases, or DNA deoxyribophosphodiesterase. Another hypothetical repair pathway hinging on a 5' --> 3' exonuclease activity is unlikely since RE is approximately 0.3 in cells deficient in either the 5' --> 3' exonuclease activities of DNA polymerase I, exonuclease VII, or RecJ. Thus, aside from NER, it is unclear what else participates in this recombination-independent repair pathway, although a pathway involing NER followed by replicative bypass of the lesion is the current working hypothesis. Psoralen interstrand cross-links appear not to be repairable by this second pathway, which may have implications for the relative cytotoxicity of interstrand cross-links from different agents.

The role of adduct site-specific mutagenesis in understanding how carcinogen-DNA adducts cause mutations: perspective, prospects and problems.

Usually, a particular mutagen/carcinogen forms adducts at many sites in DNA, making it impossible to determine which type of adduct causes which mutation and why. Adduct site-specific mutagenesis studies, in which a single adduct is built into a vector, can be used to overcome this problem. The adduct can be situated in double-stranded DNA, single-stranded DNA or in a single-stranded gap, and the benefit and concerns associated with each are addressed. An adduct site-specific study is most useful when it is compared to a mutagenesis study with its corresponding mutagen/carcinogen. Mutations induced by a particular mutagen/carcinogen can be influenced by DNA sequence context, mutagen/carcinogen dose (and other changes in conditions), level of SOS induction, cell type and other factors. Thus, it is important to match the conditions of the adduct study versus the mutagen/carcinogen study as closely as possible. DNA sequence context can profoundly affect the quantitative and qualitative pattern of adduct mutagenesis, which is addressed. In vitro studies with DNA polymerases, frameshift mutagenesis and semi-targeted mutagenesis, whereby a mutation is induced near but not at the site of the adduct, are each discussed. Finally, the relationship between structural studies on adducts and mutagenesis is considered.

Targeted A --> T and G --> T mutations induced by site-specific deoxyadenosine and deoxyguanosine adducts, respectively, from the (+)-anti-diol epoxide of dibenz[a,j]anthracene in M13mp7L2.

The studies described in this report directly examined the mutagenicity in Escherichia coli of both a deoxyadenosine (dAdo) and a deoxyguanosine (dGuo) adduct derived from (+)-anti-dibenz[a,j]-anthracene-3,4-diol 1,2-epoxide [(+)anti-DB[a,j]A-DE] that were site-specifically placed in a single-stranded M13mp7L2 replication vector. An 11-base oligonucleotide (5'-CTC ACG CTT CT-3') containing either a single (+)anti-DB[a,j]A-DE--trans-N2-dGuo or (+)anti-DB[a,j]A-DE--trans-N6dAdo adduct was successfully incorporated into single-stranded M13mp7L2 plasmid via ligation. In vitro studies using E. coli DNA polymerase I (Klenow fragment)indicated that both adducts were effective blocks for polymerase action. E. coli strains JM103 and JM103 uvrA6 were subsequently transformed with control (unadducted) and adduct-containing M13mp7L2 constructs followed by analysis of progeny DNA. In both JM103 and JM103 uvrA6 cells, plaque yields were markedly reduced with adduct containing vectors compared to control vectors. Activation of the inducible bacterial DNA repair system (SOS) by UV light only slightly increased the number of plaques recovered from either bacterial strain transformed with adduct-containing vectors. Targeted mutations were obtained with both adduct-containing vectors in both bacterial strains, whereas no mutations were detected in plaques recovered from control M13mp7L2 vectors. In JM103 cells, (+)anti-DB[a,j]A-DE--N6-dAdo induced exclusively A --> t transversions and (+)anti-DB[a,j]A-DE--N2-dGuo induced exclusively G --> T transversions. In JM103 uvrA6 cells, similar targeted transversion mutations were also obtained except that a few C deletions (i.e., aprroximately 10% of the mutations) were detected immediately 3' to the dAdo adduct. While mutagenesis was SOS dependent in JM103 cells [<0.15% (-SOS) vs approximately 1.3% (+SOS)], it appeared to be SOS independent in JM103 uvrA6 cells (approximately 1-2% in the presence or absence of SOS induction). It is argued that adduct-induced G --> T mutations can be rationalized by either misinformational or noninformational mechanisms. In contrast, A --> T mutations are unlikely to arise via a misinformational pathway, which provides the strongest support to date that bulky DNA adducts can induce mutations via a noninformational pathway.

The major, N2-Gua adduct of the (+)-anti-benzo[a]pyrene diol epoxide is capable of inducing G-->A and G-->C, in addition to G-->T, mutations.

Mutations induced by the (+)-anti-diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] were collected in the supF gene of the Escherichia coli plasmid pUB3. pUB3 was reacted with (+)-anti-B[a]-PDE and then either (1) transformed immediately into E. coli or (2) heated at 80 degrees C for 10 min and then cooled prior to transformation--the latter to probe mechanism [Rodriguez & Loechler (1993) Biochemistry 32, 1759]. Qualitatively, heating did not affect the mutagenic pattern, except at the major base substitution hotspot in supF, G115, where principally G-->T mutations were obtained prior to heating, while after heating, G-->A and G-->C mutations became statistically significantly more prevalent. Several studies have suggested that a heat-induced chemical transformation of a (+)-anti-B[a]PDE adduct at G115 (e.g., into an apurinic site) is not likely to explain the change in mutational pattern. The most likely model is that (+)-anti-B[a]P-N2-Gua is initially trapped in a metastable conformation giving principally G-->T mutations, while heating induces a change to a stable conformation(s) resulting in G-->T, A, and C mutations. This suggests that adduct conformational complexity is at the root of adduct mutational complexity. To investigate this model, a plasmid (B[a]P-G115-pRE1) with (+)-anti-B[a]P-N2-Gua in the G115 sequence context is constructed using adduct site-specific techniques.(ABSTRACT TRUNCATED AT 250 WORDS)

How are potent bulky carcinogens able to induce such a diverse array of mutations?

Mutations induced by activated benzo[a]pyrene ((+)-anti-B[a]PDE) in Escherichia coli are being investigated, by using both random and adduct-site-specific mutagenesis approaches. A working hypothesis was proposed that the major adduct of (+)-anti-B[a]PDE (formed at N2-Gua) is able to induce different base-substitution mutations (e.g., GC-->TA vs. GC-->AT) depending upon its conformation in DNA, which can be influenced by various factors, notably DNA sequence context. Frameshift mutations are also common with (+)-anti-B[a]PDE, and other work suggested that the frameshift and base-substitution mutagenesis pathways are coupled. The simplest hypothesis to rationalize this interrelationship is that a single (+)-anti-B[a]PDE adduct in a single conformation can be bypassed via either a frameshift or a base-substitution pathway. This counterintuitive notion can be reconciled if there are two different kinds of conformations on the pathway to mutagenesis: a class I conformation, which is the initial conformation of a DNA adduct in double-stranded DNA before its encounter with a DNA polymerase, and a class II conformation, which is the conformation that forms at a single-strand/double-strand DNA junction during replication by a DNA polymerase. Thus, GC-->TA and GC-->AT mutations may be induced by different class I conformations, whereas base substitution and frameshift mutations may be induced by the same class I conformation but by different class II conformations. The pathway of mutagenesis would be dictated by the relevant class I and II conformations, which in turn would be controlled by various factors, notably DNA sequence context.

The major, N2-Gua adduct of the (+)-anti-benzo[a]pyrene diol epoxide can be unstable in double-stranded DNA.

The mechanisms of mutagenesis by the (+)-anti diol epoxide of benzo[a]pyrene [(+)-anti-B[a]PDE] was investigated in supF of the Escherichia coli plasmid pUB3 [Rodriguez & Loechler (1993) Biochemistry 32, 1759]. pUB3 was reacted with (+)-anti-B[a]PDE, then either (1) transformed immediately into E. coli or (2) heated at 80 degrees C for 10 min prior to transformation--the latter to probe mechanism. Qualitatively, heating did not have a statistically significant effect on the mutagenic pattern, except at the major base substitution hot spot, G115, in supF; principally, G115-->T mutations were obtained prior to heating, while after heating, G115-->A and G115-->C mutations became more prevalent. Quantitatively, heating caused an approximately 2-fold decrease in mutation frequency. Heating released a small fraction of adducts (approximately 5%), and the chemistry and implications of this reaction are investigated herein. Although the major adduct of (+)-anti-B[a]PDE (formed at N2-Gua) is generally regarded to be heat stable, it can be quite unstable in double-stranded (but not single-stranded) DNA at low [Mg2+]. Heating releases the corresponding tetraols from (+)-anti-B[a]P-N2-Gua in approximately the same ratio as for simple hydrolysis of (+)-anti-B[a]PDE itself. This and other results suggest that guanine remains in DNA when (+)-anti-B[a]P-N2-Gua adducts are hydrolyzed. [No evidence for any other chemical change in (+)-anti-B[a]PDE adducts was found.] While no general acid/base or nucleophilic catalysis was observed, adduct hydrolysis was specific acid catalyzed down to pH approximately 5.6, where the pH-rate profile showed a break to a slope of approximately 0.0. This break probably indicates the pKa of (+)-anti-B[a]P-N2-Gua protonated at the N2-position, which is higher than expected.(ABSTRACT TRUNCATED AT 250 WORDS)

Are base substitution and frameshift mutagenesis pathways interrelated? An analysis based upon studies of the frequencies and specificities of mutations induced by the (+)-anti diol epoxide of benzo[a]pyrene.

(+)-anti-B[a]PDE-induced mutagenesis is being investigated, including in a supF gene of the E. coli plasmid pUB3. Based upon various findings a working hypothesis was proposed that the major adduct of (+)-anti-B[a]PDE (formed at N2-Gua) is able to induce different base substitution mutations (e.g., GC-->TA vs. GC-->AT vs. GC-->CG) depending upon its conformation in DNA, which can be influenced by various factors, such as DNA sequence context. Frameshift mutations are also significant and are analyzed herein. In virtually all cases one of three possibilities is observed: (1) some treatments change frameshift and base substitution mutation frequency (MF) in a quantitatively parallel fashion; (2) other treatments, which change frameshift MF, can change base substitution MF in a quantitatively reciprocal fashion; finally, (3) there are treatments that do not change frameshift MF, and also do not change base substitution MF. (Changes can be brought about by SOS induction, differing DNA sequence context, or heating adducted pUB3 prior to transformation. Why different kinds of changes result in (1) vs. (2) vs. (3) is discussed.) Thus, base substitution and frameshift mutagenesis pathways appear to be coupled in some way, which is most easily rationalized if both pathways are interrelated. The simplest mechanism to rationalize this coupling is that a single (+)-anti-B[a]PDE adduct in a single conformation can be bypassed via either a frameshift or a base substitution pathway. The surprising implication is that--although different conformations are likely to be required to induce different base substitution mutations (e.g., GC-->TA vs. GC-->AT; see above)--a single conformation can give rise to either a base substitution or a frameshift mutation. Frameshift and base substitution pathways must eventually diverge, and it is proposed that this is controlled by factors such as DNA sequence context.