Mutagenesis by site-specific arylamine adducts in plasmid DNA: enhancing replication of the adducted strand alters mutation frequency

Site specifically modified plasmids were used to determine the mutagenic effects of single arylamine adducts in bacterial cells. A synthetic heptadecamer bearing a single N-(guanin-8-yl)-2-aminofluorene (AF) or N-(guanin-8-yl)-2-(acetylamino)fluorene (AAF) adduct was used to introduce the adducts into a specific site in plasmid DNA that contained a 17-base single-stranded region complementary to the modified oligonucleotide. Following transformation of bacterial cells with the adduct-bearing DNA, putative mutants were detected by colony hybridization techniques that allowed unbiased detection of all mutations at or near the site of the adduct. The site-specific AF or AAF adducts were also placed into plasmid DNA that contained uracil residues on the strand opposite that bearing the lesions. The presence of uracil in one strand of the DNA decreases the ability of the bacterial replication system to use the uracil-containing strand, thereby favoring the use of the strand bearing the adducts. In a comparison of the results obtained with site specifically modified DNA, either with or without uracil, the presence of the uracil increased the mutation frequencies of the AF adduct by greater than 7-fold to 2.9% and of the AAF adduct by greater than 12-fold to 0.75%. The mutation frequency of the AF adduct was greatly reduced in a uvrA- strain while no mutations occurred with the AAF adduct in this strain. The sequence changes resulting from these treatments were dependent on adduct structure and the presence or absence of uracil on the strand opposite the adducts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenesis by single site-specific arylamine-DNA adducts. Induction of mutations at multiple sites

Two related carcinogen adducts, N-(deoxyguanosin-8-yl)-2-aminofluorene (AF) or N-(deoxyguanosin-8-yl)-N-acetyl-2-aminofluorene (AAF), were introduced into the lacZ' gene at base position 6253 of the minus strand of M13mp9 viral DNA. The construction of this site-specifically modified DNA was accomplished by first preparing a gapped heteroduplex missing 7 nucleotides at position 6251-6257 followed by ligation with an unmodified heptamer or with a heptamer containing either an AF or AAF adduct. These site-specifically modified templates were transfected into competent wild-type Escherichia coli cells (JM103) and a uvrA strain (SMH12). The mutation spectrum was determined by phenotypic selection of colorless plaques indicating a defective beta-galactosidase marker enzyme and by an in situ hybridization procedure to detect single base pair mismatches in the adduct region. DNA sequencing was used to characterize 179 of the mutants obtained. We found that both adducts were capable of inducing base substitution mutations at the adduct site and in the local region of the adduct. A specific frameshift (+1G) was also observed at a displaced site. All of the frameshift mutations occurred at the ligation site of the modified oligonucleotide. Control experiments with an unmodified oligonucleotide did not show an enhancement of mutations at this site, indicating that the adducts may have been responsible for these frameshifts. The mutations spectra induced by these adducts suggest that mutagenesis depends not only on adduct structure but also the sequence in which the adduct is located and the host cell type used for mutation expression.     

The role of specific amino acid residues in the active site of Escherichia coli DNA polymerase I on translesion DNA synthesis across from and past an N-2-aminofluorene adduct

Understanding how carcinogenic DNA adducts compromise accurate DNA replication is an important goal in cancer research. A central part of these studies is to determine the molecular mechanism that allows a DNA polymerase to incorporate a nucleotide across from and past a bulky adduct in a DNA template. To address the importance of polymerase architecture on replication across from this type of bulky DNA adduct, three active-site mutants of Escherichia coli DNA polymerase I (Klenow fragment) were used to study DNA synthesis on DNA modified with the carcinogen N-2-aminofluorene (AF). Running-start synthesis studies showed that full-length synthesis past the AF adduct was inhibited for all of the mutants, but that this inhibition was substantially less for the F762A mutant. Single nucleotide extension and steady-state kinetic experiments showed that the Y766S mutant displayed higher rates of insertion of each incorrect nucleotide relative to WT across from the dG-AF adduct. This effect was not observed for F762A or E710A mutants. Similar experiments that measured synthesis one nucleotide past the dG-AF adduct revealed an enhanced preference by the F762A mutant for dG opposite the T at this position. Finally, synthesis at the +1 and +2 positions was inhibited to a greater extent for the Y766S and E710A mutants compared with both the WT and F762A mutants. Taken together, this work is consistent with the model that polymerase geometry plays a crucial role in both the insertion and extension steps during replication across from bulky DNA lesions.     

Mechanism for N-acetyl-2-aminofluorene-induced frameshift mutagenesis by Escherichia coli DNA polymerase I (Klenow fragment)

N-Acetyl-2-aminofluorene (AAF) is a chemical carcinogen that reacts with guanines at the C8 position in DNA to form a structure that interferes with DNA replication. In bacteria, the NarI restriction enzyme recognition sequence (G1G2CG3CC) is a very strong mutational hot spot when an AAF adduct is positioned at G3 of this sequence, causing predominantly a -2 frameshift GC dinucleotide deletion mutation. In this study, templates were constructed that contained an AAF adduct at this position, and primers of different lengths were prepared such that the primer ended one nucleotide before or opposite or one nucleotide after the adduct site. Primer extension and gel shift binding assays were used to study the mechanism of bypass by the Escherichia coli DNA polymerase I (Klenow fragment) in the presence of these templates. Primer extension in the presence of all four dNTPs produced a fully extended product using the unmodified template, while with the AAF-modified template synthesis initially stalled at the adduct site and subsequent synthesis resulted in a product that contained the GC dinucleotide deletion. Extension product and gel shift binding analyses were consistent with the formation of a two-nucleotide bulge structure upstream of the active site of the polymerase after a nucleotide is incorporated across from the adduct. These data support a model in which the AAF adduct in the NarI sequence specifically induces a structure upstream of the polymerase active site that leads to the GC frameshift mutation and that it is this structure that allows synthesis past the adduct to occur.     

Mechanistic insights into replication across from bulky DNA adducts: a mutant polymerase I allows an N-acetyl-2-aminofluorene adduct to be accommodated during DNA synthesis

The molecular mechanism that allows a polymerase to incorporate a nucleotide opposite a DNA lesion is not well-understood. One way to study this process is to characterize the altered molecular interactions that occur between the polymerase and a damaged template. Prior studies have determined the polymerase-template dissociation constants and used kinetic analyses and a protease digestion assay to measure the effect of various DNA adducts positioned in the active site of Klenow fragment (KF). Here, a mutator polymerase was used in which the tyrosine at position 766 of the KF has been replaced with a serine. This position is located at the junction of the fingers and palm domain and is thought to be involved in maintaining the active site geometry. The primer-template was modified with N-acetyl-2-aminofluorene (AAF), a well-studied carcinogenic adduct. The mutant polymerase displayed a significant increase in the rate of incorporation of the correct nucleotide opposite the adduct but was much less prone to incorporate an incorrect nucleotide relative to the wild-type polymerase. Both the wild-type and the mutant polymerase bound much more tightly to the AAF-modified primer-template; however, unlike the wild-type polymerase, the binding strength of the mutant was influenced by the presence of a dNTP. Moreover, the mutant polymerase was able to undergo a dNTP-induced conformational change when the AAF adduct was positioned in the active site, while the wild-type enzyme could not. A model is proposed in which the looser active site of the mutant is able to better accommodate the AAF adduct.     

Recognition and repair of 2-aminofluorene- and 2-(acetylamino)fluorene-DNA adducts by UVRABC nuclease

Recognition of damage induced by N-hydroxy-2-aminofluorene (N-OH-AF) and N-acetoxy-2-(acetylamino)fluorene (NAAAF) in both phi X174 RFI supercoiled DNA and a linear DNA fragment by purified UVRA, UVRB, and UVRC proteins was investigated. We have previously demonstrated that N-OH-AF and NAAAF treatments produce N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) and N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-C8-AAF), respectively, in DNA. Using a piperidine cleavage method and DNA sequence analysis, we have found that all guanine residues can be modified by N-OH-AF and NAAAF. These two kinds of adducts have different impacts on the DNA helix structure; while dG-C8-AF maintains the anti configuration, dG-C8-AAF is in the syn form. phi X174 RF DNA-Escherichia coli transfection results indicate that while the uvrA, uvrB, and uvrC gene products are needed to repair dG-C8-AAF, the uvrC, but not the uvrA or uvrB gene products, is needed for repair of dG-C8-AF. However, we have found that in vitro the UVRA, UVRB, and UVRC proteins must work in concert to nick both dG-C8-AF and dG-C8-AAF. In general, the reactions of UVRABC nuclease toward dG-C8-AF are similar to those toward dG-C8-AAF; it incises seven to eight nucleotides from the 5' side and three to four nucleotides from the 3' side of the DNA adduct. Evidence is presented to suggest that hydrolysis on the 3' and 5' sides of the damaged base by UVRABC nuclease is not simultaneous and that at least occasionally hydrolysis occurs only on the 3' side or on the 5' side of the damage site. The possible mechanisms of UVRABC nuclease incision for AF-DNA are discussed.     

Observing translesion synthesis of an aromatic amine DNA adduct by a high-fidelity DNA polymerase

Aromatic amines have been studied for more than a half-century as model carcinogens representing a class of chemicals that form bulky adducts to the C8 position of guanine in DNA. Among these guanine adducts, the N-(2'-deoxyguanosin-8-yl)-aminofluorene (G-AF) and N-2-(2'-deoxyguanosin-8-yl)-acetylaminofluorene (G-AAF) derivatives are the best studied. Although G-AF and G-AAF differ by only an acetyl group, they exert different effects on DNA replication by replicative and high-fidelity DNA polymerases. Translesion synthesis of G-AF is achieved with high-fidelity polymerases, whereas replication of G-AAF requires specialized bypass polymerases. Here we have presented structures of G-AF as it undergoes one round of accurate replication by a high-fidelity DNA polymerase. Nucleotide incorporation opposite G-AF is achieved in solution and in the crystal, revealing how the polymerase accommodates and replicates past G-AF, but not G-AAF. Like an unmodified guanine, G-AF adopts a conformation that allows it to form Watson-Crick hydrogen bonds with an opposing cytosine that results in protrusion of the bulky fluorene moiety into the major groove. Although incorporation opposite G-AF is observed, the C:G-AF base pair induces distortions to the polymerase active site that slow translesion synthesis.     

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

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

Preparation and characterization of a viral DNA molecule containing a site-specific 2-aminofluorene adduct: a new probe for mutagenesis by carcinogens

The synthetic oligonucleotide heptamer 5'-ATCCGTC-3' was reacted in vitro with N-acetoxy-N-(trifluoroacetyl)-2-aminofluorene and the resulting product isolated by reverse-phase high-performance liquid chromatography (HPLC). This purified oligonucleotide, which was shown by chemical and enzymatic analysis to be a heptamer containing a single N-(deoxyguanin-8-yl)-2-aminofluorene adduct, was then used to situate the putatively mutagenic aminofluorene lesion within the genome of M13 mp9 by ligating it into a complementary single-stranded region located at a specific site in the negative strand of the duplex M13 mp9 DNA molecule. The presence of the adduct at the anticipated location was confirmed by taking advantage of the facts that AF adducts inhibit many restriction enzymes when located in or near their restriction sites and that the AF moiety should be contained within the HincII recognition sequence on M13 mp9 DNA. Upon attempted cleavage of the M13 DNA containing the site-specific AF adduct with HincII, we find that the large majority of the DNA remained circular, demonstrating the incorporation of the AF adduct in high yield into the DNA molecule at this location. This system should prove useful in vivo for the study of mutagenesis by chemical carcinogens and in vitro to study the interaction of purified DNA metabolizing proteins with a template containing a site-specific lesion.     

Recognition and incision of site-specifically modified C8 guanine adducts formed by 2-aminofluorene, N-acetyl-2-aminofluorene and 1-nitropyrene by UvrABC nuclease

Nucleotide excision repair plays a crucial role in removing many types of DNA adducts formed by UV light and chemical carcinogens. We have examined the interactions of Escherichia coli UvrABC nuclease proteins with three site-specific C8 guanine adducts formed by the carcinogens 2-aminofluorene (AF), N-acetyl-2-acetylaminofluorene (AAF) and 1-nitropyrene (1-NP) in a 50mer oligonucleotide. Similar to the AF and AAF adducts, the 1-NP-induced DNA adduct contains an aminopyrene (AP) moiety covalently linked to the C8 position of guanine. The dissociation constants for UvrA binding to AF–, AAF– and AP–DNA adducts, determined by gel mobility shift assay, are 33 ± 9, 8 ± 2 and 23 ± 9 nM, respectively, indicating that the AAF adduct is recognized much more efficiently than the other two. Incision by UvrABC nuclease showed that AAF–DNA was cleaved ~2-fold more efficiently than AF– or AP–DNA (AAF > AF ≈ AP), even though AP has the largest molecular size in this group. However, an opened DNA structure of six bases around the adduct increased the incision efficiency for AF–DNA (but not for AP–DNA), making it equivalent to that for AAF–DNA. These results are consistent with a model in which DNA damage recognition by the E.coli nucleotide excision repair system consists of two sequential steps. It includes recognition of helical distortion in duplex DNA followed by recognition of the type of nucleotide chemical modification in a single-stranded region. The difference in incision efficiency between AF– and AAF–DNA adducts in normal DNA sequence, therefore, is a consequence of their difference in inducing structural distortions in DNA. The results of this study are discussed in the light of NMR solution structures of these DNA adducts.     

Age-related induction and disappearance of carcinogen-DNA-adducts in livers of rats exposed to low levels of 2-acetylaminofluorene

It was investigated whether in vivo aging of rat liver is associated with changes in the induction and rate of disappearance of DNA damage. For this purpose 6- and 36-month-old rats were intraperitoneally injected with a single, low dose (5 mg/kg body wt.) of the model liver carcinogen 2-acetylaminofluorene (AAF). Using the 32P-postlabeling assay we found that N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) was the major DNA-adduct formed. The minor adduct N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) could only be detected after doses of 20 mg/kg or more. Quantitation of adduct levels at various time points after treatment indicated a rapid induction of AF-adducts, which were already present at 6 h after treatment. The subsequent loss of AF-adducts was relatively slow, as was indicated by the presence of a substantial amount of AF-adducts as late as 21 days after treatment. Slight age-related differences in the pattern of induction and disappearance of AF-adducts and a somewhat higher level of persisting lesions in old than in young rats were observed.     

Differential effects induced by N-hydroxy-2-acetylaminofluorene and N-hydroxy-2-aminofluorene in DNA excision-repair-deficient Chinese hamster cells

The direct-acting cytotoxic properties of N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxy-2-aminofluorene (N-OH-AF) have been determined in repair-proficient (AA8-4) and repair-deficient (UV-5) Chinese hamster ovary cells. Cytotoxicity comparisons indicate that UV-5 cells are considerably more sensitive to exposure to N-OH-AAF than is the parental AA8-4 cell line, i.e., concentrations needed to obtain a D37 for survival of AA8-4 is greater than 5-fold higher than for UV-5. Mutation analysis at the HGPRT locus also indicates the increased sensitivity of UV-5 cells to N-OH-AAF as witnessed by an enhanced induction of 6-thioguanine-resistant colonies at equitoxic doses. Conversely, N-OH-AAF, did not induce a 'UV-mimetic' response when comparing genotoxicity between these two cell lines. Our data coupled with previously published model-building and adduct removal studies (Broyde and Hingerty, 1983; Fuchs and Daune, 1974; Grunberger and Weinstein, 1976; Yamasaki et al., 1977) suggest that the minor DNA adduct species, N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene, may be responsible for the hypermutagenicity witnessed in DNA excision-repair-deficient cells treated with N-OH-AAF.     

Conformational and thermodynamic properties modulate the nucleotide excision repair of 2-aminofluorene and 2-acetylaminofluorene dG adducts in the NarI sequence

Nucleotide excision repair (NER) is a major repair pathway that recognizes and corrects various lesions in cellular DNA. We hypothesize that damage recognition is an initial step in NER that senses conformational anomalies in the DNA caused by lesions. We prepared three DNA duplexes containing the carcinogen adduct N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-acetylaminofluorene (FAAF) at G(1), G(2) or G(3) of NarI sequence (5'-CCG(1)G(2)CG(3)CC-3'). Our (19)F-NMR/ICD results showed that FAAF at G(1) and G(3) prefer syn S- and W-conformers, whereas anti B-conformer was predominant for G(2). We found that the repair of FAAF occurs in a conformation-specific manner, i.e. the highly S/W-conformeric G(3) and -G(1) duplexes incised more efficiently than the B-type G(2) duplex (G(3)～G(1)> G(2)). The melting and thermodynamic data indicate that the S- and W-conformers produce greater DNA distortion and thermodynamic destabilization. The N-deacetylated N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene (FAF) adducts in the same NarI sequence are repaired 2- to 3-fold less than FAAF: however, the incision efficiency was in order of G(2)～G(1)> G(3), a reverse trend of the FAAF case. We have envisioned the so-called N-acetyl factor as it could raise conformational barriers of FAAF versus FAF. The present results provide valuable conformational insight into the sequence-dependent UvrABC incisions of the bulky aminofluorene DNA adducts.     

Carcinogenic adducts induce distinct DNA polymerase binding orientations

DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism.     

Energy minimized structures of carcinogen-DNA adducts: 2-acetylaminofluorene and 2-aminofluorene

Energy minimized structures of DNA modified by the aromatic amines 2-acetylaminofluorene (AAF) and 2-aminofluorene (AF), for which no experimental atomic resolution data exist, are presented. These have been computed with a new molecular mechanics program specifically designed to define distortions imposed by such adducts, and employing a rational strategy for searching the conformation space of a DNA molecule with covalently linked carcinogen. In alternating G-C sequences, the AAF adduct prefers to reside at the exterior of an undeformed Z-helix. It can also induce base displacement with attendant denaturation and helix bending in sequences that disfavor the Z form, but undeformed B helices are excluded. The AF adduct, by contrast, prefers the major groove of an unperturbed B-helix, but can also induce carcinogen-base stacking in single stranded regions of the DNA, such as at the replication fork. The different biological properties of these two adducts may be related to their distinct     

In situ detection of acetylaminofluorene-DNA adducts in human cells using monoclonal antibodies

The present study was performed to generate monoclonal antibodies capable of detecting N-acetoxy-2-acetylaminofluorene (NA-AAF)-derived DNA adducts in human cells in situ. As an immunogen, we employed NA-AAF-modified single-stranded DNA coupled electrostatically to methylated protein and we produced five different monoclonal antibodies. All of them showed strong binding to NA-AAF-modified DNA, but had undetectable or minimal binding to undamaged DNA. Competitive inhibition experiments revealed that the epitope recognized by these antibodies is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-C8-AAF) in DNA, although deacetylated N-(deoxyguanosin-8-yl)-2-aminofluorene in DNA is also recognized with slightly less efficiency. In contrast, these antibodies did not bind to 3-(deoxyguanosin-N(2)-yl)-2-acetylaminofluorene in DNA or to UV-induced lesions in DNA. Interestingly, they showed only minimal binding to small AAF-nucleoside adducts (dG-C8-AAF), indicating that DNA regions flanking a DNA-bound adduct, in addition to the adduct itself, are essential for the stable binding of the antibodies. Using an enzyme-linked immunosorbent assay with the most promising antibody (AAF-1), we detected the concentration-dependent induction of NA-AAF-modified adducts in DNA from repair deficient xeroderma pigmentosum (XP) cells treated with physiological concentrations of NA-AAF. Moreover, the assay enabled to confirm that normal human cells efficiently repaired NA-AAF-induced DNA adducts but not XP-A cells. Most importantly, the formation of NA-AAF-induced DNA adducts in individual nuclei of XP cells could be clearly visualized using indirect immunofluorescence. Thus, we succeeded in establishing novel monoclonal antibodies capable of the in situ detection of NA-AAF-induced DNA adducts in human cells.     

The formation of 2-aminofluorene-DNA adducts in vivo: evidence for peroxidase-mediated activation

Formation of DNA adducts in various tissues of dogs fed a single dose of the carcinogen 2-aminofluorene was investigated. Adduct analysis was performed using a technique that allows measurement of both N-(deoxyguanosin-8-yl)-2-amino-2-aminofluorene-DNA adduct formed by reaction of N-hydroxy-2-aminofluorene with DNA, as well as the polar 2-aminofluorene-DNA adducts formed when 2-aminofluorene is activated by prostaglandin H synthase-peroxidase in vitro. Two male beagle (A and B) dogs were examined and a different DNA adduct profile was observed with each dog. For the dog A, N-(deoxyguanosin-8-yl)-2-aminofluorene was the major adduct found in hepatic DNA; no peroxidase-derived adducts were detected in this tissue. In contrast, adducts eluting similarly to peroxidase-derived adducts were found in urinary tract tissues of this dog with the relative abundance of these adducts in the order urothelium greater than renal medulla greater than renal cortex, which correlates with the respective tissues' prostaglandin H synthase activity. N-(Deoxyguanosin-8-yl)-2-aminofluorene was detected in the renal tissues, but not in urothelium. For dog B, only the N-(deoxyguanosin-8-yl)-2-aminofluorene adduct was observed in all tissues examined, including the urothelium. However, total binding to liver, kidney, and bladder were two-, two-, and four-fold lower, respectively, than dog A. These data indicate that both prostaglandin H synthase-mediated activation and N-hydroxylation of 2-aminofluorene occur in vivo and may be subjected to pharmacodynamic considerations. Furthermore, the tissue distribution of the peroxidase-mediated 2-aminofluorene adducts suggests this process may also be of importance in the bladder-specific carcinogenicity of aromatic amines.     

Probing the sequence effects on NarI-induced -2 frameshift mutagenesis by dynamic 19F NMR, UV, and CD spectroscopy

The NarI recognition sequence (5'-G1G2CG3CN-3') is the most vulnerable hot spot for frameshift mutagenesis induced by the carcinogen 2-aminofluorene and its analogues in Escherichia coli. Lesioning of the guanine in the G3 position induces an especially high frequency of -2 deletion mutations; vulnerability to these mutations is modulated by the nature of the nucleotide in the N position (C approximately A > G > T). The objective of the present study was to probe the structural basis of this N-mediated influence on the propensity of the G3 lesion to form a slipped mutagenic intermediate (SMI) during translesion synthesis. We studied NarI-based fully paired [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNCGGCCGAG-3'), N = dC or dT] and -2 deletion [(5'-CTCG1G2CG3*CNATC-3')(5'-GATNGCCGAG-3'), N = dC or dT] duplexes, in which G* was either AF [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene] or the 19F probe FAF [N-(2'-deoxyguanosin-8-yl)-7-fluoro-2-aminofluorene]. The latter sequences mimic the bulged SMI for -2 deletion mutations. Dynamic 19F NMR, circular dichroism, and UV melting results indicated that the NarI-dC/-2 deletion duplex adopts exclusively an intercalated conformer, whereas the NarI-dT/-2 deletion duplex exists as multiple conformers. The data support the presence of a putative equilibrium between a carcinogen-intercalated and a carcinogen-exposed SMI for the dT/-2 duplex. A similar dT-induced conformational heterogeneity was observed for the fully paired duplexes in which all three guanines were individually modified by AF or FAF. The frequency of the carcinogen stacked S-conformation was found to be highest (69-75%) at the G3 hot spot in NarI-dC duplexes. Taken together, our results support the hypothesis that the conformational stability of the SMI is a critical determinant for the efficacy of -2 frameshift mutagenesis in the NarI sequence. We also provide evidence for AF/FAF conformational compatibility in the NarI sequences.     

Probing the conformational heterogeneity of the acetylaminofluorene-modified 2'-deoxyguanosine and DNA by 19F NMR spectroscopy.

19F NMR spectroscopy was used to probe the conformation of a DNA adduct derived from the carcinogen 7-fluoro-N-acetyl-2-aminofluorene (FAAF) in three structural contexts: as a monomer and incorporated into single- and double-stranded DNA. The 19F NMR spectrum of dG-C8-FAAF [N-(deoxyguanosin-8-yl)-N-acetyl-7-fluoro-2-aminofluorene] in methanol at -30 degrees C exhibited four interconvertible signals in a 11:52:26:11 ratio. Dynamic NMR analysis indicated that the four torsional isomers arise from restricted rotation about the amide (gamma) (14.4 kcal/mol) and the guanyl-nitrogen (alpha) bonds. The conformational heterogeneity persisted in a single strand FAAF-12-mer, d(CTTCTTG[FAAF]ACCTC), whose 19F NMR spectrum at 22 degrees C and pH 7.0 gave only two signals in a 40:60 ratio, instead of four. The two 19F signals followed a two-site exchange with the rotation barrier of 14.7 kcal/mol about the amide (gamma') bond. A similar conformational theme was observed in the FAAF-12-mer duplex, d(CTTCTTG[FAAF]ACCTC).d(GAGGTCAAGAAG), which revealed two 19F resonances in a 41:59 ratio at 22 degrees C and pH 7.0. According to solvent-induced isotope and magnetic anisotropy effects, the two duplex conformers adopt exclusively a base displacement structure, being different only in their relative acetyl group orientations, cis (gamma' approximately 180 degrees) or trans (gamma' approximately 0 degrees ). Dynamic NMR data indicated that the two conformers do not exchange over a wide range of temperatures. This contrasts with the nonacetylated counterpart, which exhibits an equilibrium between the "B-type" and "stacked" conformers [Zhou, L., et al. (1997) J. Am. Chem. Soc. 119, 5384-5389]. The exclusive stacked nature of the AAF adducts may provide insight into why AAF adducts are more mutagenic and prone to repair than the nonacetylated AF adducts.     

Examination of the long-range effects of aminofluorene-induced conformational heterogeneity and its relevance to the mechanism of translesional DNA synthesis

Adduct-induced conformational heterogeneity complicates the understanding of how DNA adducts exert mutation. A case in point is the N-deacetylated AF lesion [N-(2'-deoxyguanosin-8-yl)-2-aminofluorene], the major adduct derived from the strong liver carcinogen N-acetyl-2-aminofluorene. Three conformational families have been previously characterized and are dependent on the positioning of the aminofluorene rings: B is in the "B-DNA" major groove, S is "stacked" into the helix with base-displacement, and W is "wedged" into the minor groove. Here, we conducted (19)F NMR, CD, T(m), and modeling experiments at various primer positions with respect to a template modified by a fluorine tagged AF-adduct (FAF). In the first set, the FAF-G was paired with C and in the second set it was paired with A. The FAF-G:C oligonucleotides were found to preferentially adopt the B or S-conformers while the FAF-G:A mismatch ones preferred the B and W-conformers. The conformational preferences of both series were dependent on temperature and complementary strand length; the largest differences in conformation were displayed at lower temperatures. The CD and T(m) results are in general agreement with the NMR data. Molecular modeling indicated that the aminofluorene moiety in the minor groove of the W-conformer would impose a steric clash with the tight-packing amino acid residues on the DNA binding area of the Bacillus fragment (BF), a replicative DNA polymerase. In the case of the B-type conformer, the carcinogenic moiety resides in the solvent-exposed major groove throughout the replication/translocation process. The present dynamic NMR results, combined with previous primer extension kinetic data by Miller & Grollman, support a model in which adduct-induced conformational heterogeneities at positions remote from the replication fork affect polymerase function through a long-range DNA-protein interaction.