Effects of benzo[a]pyrene DNA adducts on Escherichia coli DNA polymerase I (Klenow fragment) primer-template interactions: evidence for inhibition of the catalytically active ternary complex formation.

Benzo[a]pyrene diol epoxide (B[a]PDE) adducts are strong blocks of DNA replication in vitro, allowing the rare incorporation of a nucleotide across from the lesion and negligibly small extent of further bypass. To study the mechanistic details of this process, a gel-retardation assay was used to measure the dissociation constants for the binding of DNA polymerase I (Klenow fragment) (KF) to the primer-templates containing a (+)-trans- or (+)-cis-B[a]P-N(2)-dG adduct. When the primer was terminated one nucleotide before the adduct, the presence of a (+)-trans-B[a]P-N(2)-dG adduct did not affect the binding while a (+)-cis-B[a]P-N(2)-dG adduct caused a slight decrease in affinity. The presence of any dNTP decreased the affinity of KF to the modified primer-templates. (In contrast, a strong increase of the affinity to unmodified primer-templates was observed in the presence of the next correct dNTP.) Limited protease digestion experiments indicated that a closed ternary complex of KF with the modified primer-templates was not detectable in the presence of any dNTP, whereas it was clearly observed with unmodified template in the presence of the next correct nucleotide. These findings suggest that these adducts may interfere with the conformational change to the catalytically active closed ternary complex and/or cause significant destabilization of this complex. When the primers extended to the position across from the adduct, the affinity of KF was significantly decreased irrespective of the identity of the base across from the adduct, possibly explaining the low bypass of the lesion. Interestingly, the stability of these DNA-polymerase complexes correlated with nucleotide insertion kinetics for the unmodified and (+)-trans-B[a]PDE-modified templates.

Significance of nucleobase shape complementarity and hydrogen bonding in the formation and stability of the closed polymerase-DNA complex.

DNA polymerases insert a dNTP by a multistep mechanism that involves a conformational rearrangement from an open to a closed ternary complex, a process that positions the incoming dNTP in the proper orientation for phosphodiester bond formation. In this work, the importance and relative contribution of hydrogen-bonding interactions and the geometric shape of the base pair that forms during this process were studied using Escherichia coli DNA polymerase I (Klenow fragment, 3'-exonuclease deficient) and natural dNTPs or non-hydrogen-bonding dNTP analogues. Both the geometric fit of the incoming nucleotide and its ability to form Watson-Crick hydrogen bonds with the template were found to contribute to the stability of the closed ternary complex. Although the formation of a closed complex in the presence of a non-hydrogen-bonding nucleotide analogue could be detected by limited proteolysis analysis, a comparison of the stabilities of the ternary complexes indicated that hydrogen-bonding interactions between the incoming dNTP and the template increase the stability of the complex by 6-20-fold. Any deviation from the Watson-Crick base pair geometry was shown to have a destabilizing effect on the closed complex. This degree of destabilization varied from 3- to 730-fold and was found to be correlated with the size of the mismatched base pair. Finally, a stable closed complex is not formed in the presence of a ddNTP or rNTP. These results are discussed in relation to the steric exclusion model for the nucleotide insertion.

In vitro replication of primer-templates containing benzo[a]pyrene adducts by exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment): effect of sequence context on lesion bypass.

The presence of benzo[a]pyrene diol epoxide (B[a]PDE) adducts in DNA is known to interfere with DNA replication. Kinetic studies of nucleotide insertion by exonuclease-deficient E. coli DNA polymerase I (Klenow fragment) across from either the (+)-trans- or the (+)-cis-B[a]P-N(2)-dG adduct in the 5'-CGT-3' sequence context indicated that the rate of nucleotide incorporation followed the order: dAMP > dGMP > dTMP > dCMP, which did not correlate with the mutational spectrum observed for these adducts in this sequence in E. coli (mostly G-->A transitions). Interestingly, a kinetic analysis of extension past the adduct showed that, unlike other sequences studied, the primer-template was extended best when dT was positioned at the 3'-terminus of the primer across from either a (+)-trans- or a (+)-cis-B[a]P-N(2)-dG adduct. In contrast, when the (+)-trans-B[a]P-N(2)-dG adduct was positioned in the 5'-TGC-3' sequence context, which gives predominantly G-->T mutations in E. coli, extension was detectable only when dA was positioned across from the adduct. These data provide the first in vitro evidence that may explain why G-->A transitions, rather than the G-->T transversions found in other sequences, are preferred in the 5'-CGT-3' sequence in vivo.

Differential effects of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts on the conformational change in the structure of DNA polymerase I (Klenow fragment).

The carcinogen N-acetyl-2-aminofluorene forms two major DNA adducts: the N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene adduct (dG-C8-AAF) and its deacetylated derivative, the N-(2'-deoxyguanosin-8-yl)-2-aminofluorene adduct (dG-C8-AF). It is well established that the AAF adduct is a very strong block for DNA synthesis in vitro while the AF adduct is more easily bypassed. In an effort to understand the molecular mechanism of this phenomenon, the structure of the complex of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) bound to primer-templates containing either an AF or AAF adduct in or near the active site was probed by nuclease and protease digestion analyses. The results of these experiments suggest that positioning the AAF adduct in the polymerase active site strongly inhibits the conformational change that is required for the insertion of a nucleotide. Similar experiments with AF-modified primer-templates shows a much less pronounced effect. The inhibition of the conformational change by either adduct is not detected if they are positioned in the single-stranded part of the template just one nucleotide before the active site. These findings may explain the different abilities of these lesions to block DNA synthesis.

A conformational change in E. coli DNA polymerase I (Klenow fragment) is induced in the presence of a dNTP complementary to the template base in the active site.

It is well established that the insertion of a nucleotide into a growing DNA chain requires a conformational change in the structure of a DNA polymerase. These enzymes have been shown to bind a primer-template in the open conformation and then upon binding of a complementary dNTP undergo a conformational rearrangement to the closed ternary complex. This movement results in the positioning of the incoming nucleotide in the proper geometry for the nucleophilic attack by the 3'-hydroxyl of the primer. In this work, tryptic digestion experiments were performed to detect this conformational change in the structure of the exonuclease-deficient DNA polymerase I (Klenow fragment). Three distinct digestion patterns were observed: one for the polymerase alone, one for the binary complex with the primer-template, and one for the ternary polymerase-DNA-dNTP complex. The latter conformational change leads to a stable ternary closed complex formation only when the correct nucleotide is present in the reaction mixture. Positioning of nucleotides with incorrect geometry in the protein active site inhibits or eliminates formation of the closed complex. Similarly, this conformational change is inhibited when the primer terminus of the DNA molecule is altered by the presence of the 2'-hydroxyl.

Cytosine methylation in a CpG sequence leads to enhanced reactivity with Benzo[a]pyrene diol epoxide that correlates with a conformational change.

Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.

Interaction of Escherichia coli DNA polymerase I (Klenow fragment) with primer-templates containing N-acetyl-2-aminofluorene or N-2-aminofluorene adducts in the active site.

DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primer-templates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present.

Effect of mismatched complementary strands and 5'-change in sequence context on the thermodynamics and structure of benzo[a]pyrene-modified oligonucleotides.

Benzo[a]pyrene (B[a]P) is a well-studied environmental carcinogen that when activated can react with DNA to form four major adducts: (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-B[a]P-dG. In this study, two oligonucleotides (5'-dCCATT-GB[a]P-CTACC-3' and 5'-dCCATC-GB[a]P-CTACC-3') were prepared, each containing the four isomeric adducts, and these were hybridized to either complementary sequences or to sequences containing an A, G, or T opposite the adducted guanine. Thermal melting curves, CD, and UV spectra of each duplex were measured and compared with the unmodified counterpart. The raw and relative thermodynamic measurements were then compared which indicated that differences occur that are both adduct and sequence dependent. These differences were next compared with the in vitro DNA polymerase incorporation data and were found to be strikingly correlated. Most significantly, for all four B[a]P isomers a mismatch of an A across from the adduct resulted in the least amount of relative destabilization, while the Watson-Crick complement C showed the most; in vitro studies showed that A is the preferred base incorporated across from each isomer, while C was incorporated least often. This observed correlation suggests that one factor contributing to misincorporation at an adduct site is the thermodynamic stability of the incorporated base. Structurally, the effect of sequence context and mismatched complementary strands were also compared, suggesting that all adducts tend to intercalate within the helix when they are complemented with a mismatched complementary strand. In addition, the level of this intercalation seems to be both sequence and stereoisomer dependent.

Rate of incision of N-acetyl-2-aminofluorene and N-2-aminofluorene adducts by UvrABC nuclease is adduct- and sequence-specific: comparison of the rates of UvrABC nuclease incision and protein-DNA complex formation.

The UvrABC nuclease, the nucleotide excision repair complex from Escherichia coli, is able to incise a variety of types of DNA damage and the repair efficiency of this enzyme complex appears to be influenced by the structure of the damage and the sequence context within which the damage is positioned. In order to better establish these relationships, we have constructed two DNA sequences each containing a site-specifically positioned N-2-aminofluorene (AF) or N-acetyl-2-aminofluorene (AAF) adduct and have determined both the kinetics of UvrABC nuclease incision and the kinetics of UvrABC nuclease-substrate complex formation. It is well established that these two adducts induce very different structures in the DNA and that these structures also depend on the sequence context. We have found that the rate of incision of both AAF- and AF-DNA adducts is significantly faster when they are positioned in the mutation hotspot NarI sequence (5-GGCG*CC-3') than when located in a normal or non-NarI sequence (5'-GATG*ATA-3') and that the rate of incision for AAF-DNA adducts is faster that for AF adducts in both sequences. Most siginificantly, we find that the rate of UvrB and UvrBC-substrate complex formation correlates with the rate of UvrABC nuclease incision.

Human MutSalpha specifically binds to DNA containing aminofluorene and acetylaminofluorene adducts.

Defects in mismatch repair are associated with several types of cancer. It is also generally believed that environmental carcinogens are responsible for the initiation of cancers by the induction of mutations in critical genes. Prior genetic studies have suggested that the mismatch repair system can also recognize certain forms of DNA damage such as O6-methylguanine and UV photoproducts, and, therefore, mismatch repair may play a role in environmental agent-induced carcinogenesis. To examine this hypothesis, hMutSalpha, a heterodimer which consists of hMSH2 and GTBP and participates in strand-specific mismatch repair, was tested for its ability to recognize DNA containing a site-specific C8-guanine adduct of aminofluorene (AF) or N-acetyl-2-aminofluorene (AAF). We show here that hMutSalpha specifically binds to both AF and AAF adducts. This binding requires both hMSH2 and GTBP. Results from competition and titration experiments indicate that the binding efficiency of hMutSalpha to AF and AAF is about 60% of that to a G-T mismatch, but is at least 10-fold that to an otherwise identical homoduplex DNA without the chemical modification. The specific binding of AF and AAF adducts by hMutSalpha suggests that strand-specific mismatch repair is involved in processing DNA damage induced by environmental carcinogens.

Benzo[a]pyrene-DNA adducts inhibit the DNA helicase activity of the bacteriophage T7 gene 4 protein.

The gene 4 protein of bacteriophage T7 provides the essential helicase and primase activities for the replication of the T7 genome. In addition, it also displays a DNA-dependent deoxyribonucleoside triphosphatase activity, the preferred substrate of which is dTTP. Previous investigations have demonstrated that the translocation of the gene 4 protein along single-stranded DNA is blocked by the presence of benzo[a]pyrene-DNA adducts and that the gene 4 protein is likely to be sequestered at the sites of these adducts. In the present study, we directly show that the helicase activity of the gene 4 protein is also profoundly inhibited by the benzo[a]pyrene-DNA adducts. The inhibitory effects of these adducts are strand-specific in that they block the DNA helicase activity of the gene 4 protein only when they are located in the DNA strand where the gene 4 protein translocates when it unwinds double-stranded DNA. Consistent with the hypothesis that the gene 4 protein is sequestered at the adduct site, we also show that the complexes formed by the gene 4 protein and benzo[a]pyrene-modified DNA are far more stable than those formed by the gene 4 protein and unmodified DNA.

Nucleotide and DNA-induced conformational changes in the bacteriophage T7 gene 4 protein.

The bacteriophage T7 gene 4 protein is a multifunctional enzyme that has DNA helicase, primase, and deoxyribonucleotide 5'-triphosphatase activities. Prior studies have shown that in the presence of dTTP or dTDP the gene 4 protein assembles into a functionally active hexamer prior to binding to single-stranded DNA. In this study, we have examined the effects of different nucleotide cofactors on the conformation of the gene 4 protein in the presence and absence of DNA. Gel retardation analysis, partial protease digestion, and DNA footprinting all suggest that the gene 4 protein undergoes a conformational change when dTTP is hydrolyzed to dTTP and that in the presence of dTDP the complex with DNA is more open or extended. We have also found that the dissociation constant of the gene 4 protein.DNA complex in the presence of dTDP was 10-fold lower than that determined in the presence of dTTP, further suggesting that these cofactors exerts different allosteric effects on the DNA-binding site of the gene 4 protein.

A method for the purification of oligonucleotides containing strong intra- or intermolecular interactions by reversed-phase high-performance liquid chromatography.

Synthetic oligodeoxyribonucleotides containing a high guanine content have a tendency to form intra- or intermolecular complexes in solution make HPLC purification difficult or sometimes impossible. We have developed a simple method that has enabled us to purify a series of highly guanine-rich and self-complementary oligonucleotides by HPLC on a reverse-phase PRP-1 column. Although others have shown that this type of oligonucleotide can be purified on an ion-exchange column by adding formamide to the mobile phase, the resulting resolution is poor and the formamide must subsequently be removed from the purified product. We find that simply having 20% formamide in the loading buffer is sufficient to remove the interfering interactions. This small amount of formamide passes quickly through the reverse-phase column, far removed the peak position of the oligonucleotides. Quantities of up to 35 ODs have been satisfactorily purified with recoveries of 95% or better. This procedure was particularly suitable for purification of oligonucleotides containing base-labile modifications, such as acetylaminofluorene-modified oligonucleotides,since other denaturing HPLC purification methods usually employ strong alkaline conditions or high temperatures that might result in damage to the adduct.

Differences in the mechanism of stimulation of T7 DNA polymerase by two binding modes of Escherichia coli single-stranded DNA-binding protein.

Escherichia coli single-stranded DNA-binding protein (Eco SSB) has been shown previously to display several DNA binding modes depending on the ionic conditions. To determine what effect these various binding modes have on DNA replication, we have studied DNA synthesis by the T7 DNA polymerase under ionic conditions where Eco SSB interacts with either 72 or 91 nucleotides of M13 DNA. These forms presumably correspond to the previously described (SSB)56 and (SSB)65 (Lohman and Ferrari, 1994) that were determined using the binding of SSB to homopolymers. Here we report the stimulation induced by (SSB)91 to be 4-fold greater than that produced by (SSB)72 under conditions where the template is in large excess. Surprisingly, when the polymerase level is raised so that it is in molecular excess, (SSB)91 no longer stimulates synthesis while (SSB)72 affords a 4-fold stimulation, which is the same level of stimulation as when the template was in excess. Both SSB forms increase the rate of DNA synthesis and were found to stimulate synthesis by relieving template secondary structures. However, (SSB)72 specifically increases strand displacement synthesis, while (SSB)91 stimulates synthesis by increasing the affinity of the polymerase for the template.

Mutagenesis at a site-specifically modified NarI sequence by acetylated and deacetylated aminofluorene adducts.

A hotspot for mutagenesis by N-acetyl-2-aminofluorene (AAF) was site-specifically modified with 2-aminofluorene (AF) and AAF adducts, and the mutation frequencies and specificities were determined and compared. Previous work has shown that the presence of an AAF adduct in a NarI sequence (GGCGCC) results a high mutation frequency for a CG double base pair deletion. In the present study, an M13 derivative was constructed that contained a NarI recognition sequence in the beta-galactosidase gene of bacteriophage M13mp9. This derivative was site-specifically modified with either an AF or an AAF adduct, the products were characterized, and these templates were then transformed into Escherichia coli wild-type strain JM103 or uvrA strain SMH12. The levels and mutation spectra were determined either with or without SOS induction. It was found that, with SOS functions induced, the measured mutation frequencies were substantially higher in all cases. More importantly, the types of mutations induced by the AAF and AF adducts were very different: AAF adducts induced almost exclusively CG double base deletion mutations, whereas AF adducts gave rise specifically to base-substitution mutations. The AF-derived mutation spectrum included both G to T and G to A mutations. The results are discussed in light of the current views on the relationship between the DNA structure and mutagenesis.

Solid-phase synthesis of oligonucleotides containing site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetylamino)fluorene adducts using 9-fluorenylmethoxycarbonyl as the base-protecting group.

Eight oligodeoxyribonucleotides containing a site-specific N-(2'-deoxyguanosin-8-yl)-2-(acetyl-amino)fluorene (dG-C8-AAF) adduct were prepared successfully by solid-phase DNA synthesis using the 2-cyanoethyl N,N-diisopropylphosphoramidites of dA, dC, dG, dT, and dG-C8-AAF, with 9-fluorenyl-methoxycarbonyl (Fmoc) as the base-protecting group. The oligonucleotides were deprotected and released from the support by 1:9 piperidine/MeOH at room temperature for 22-36 h or by 1:1 diisopropylamine in MeOH at 55 degrees C for 15 h, purified by HPLC, and fully characterized. About 6 mg of HPLC-purified d[GTGGCG(C8-AAF)CCAAGT] and 7 mg of d[GTGATG(C8-AAF)ATAAGT] were obtained from the 10-mumol-scale synthesis, and their 1D 1H NMR spectra were consistent with the presence of a dG-C8-AAF adduct. The dG-C8-AAF oligonucleotides were also deacetylated to afford the corresponding dG-C8-AF oligonucleotides. d[GTGGCG(C8-AAF)CCAAGT] formed stable 1:1 duplexes with both the fully complementary 12-mer and a GC-deleted (across the adduct) 10-mer complement, and identical melting temperatures were observed for both duplexes. The multidimensional NMR study of these duplexes is presently under investigation.

Accurate in vitro translesion synthesis by Escherichia coli DNA polymerase I (large fragment) on a site-specific, aminofluorene-modified oligonucleotide.

We have measured the accuracy of in vitro synthesis by DNA polymerase I (large fragment) during translesion synthesis past an aminofluorene (AF) adduct. These studies were carried out using a site-specifically modified template which contained a single AF adduct. The template was prepared by first modifying the lone guanine in a 17 base long oligonucleotide and extensively purifying and characterizing this product. The modified 17mer was then ligated to a synthetic duplex to produce a 31 nucleotide long template strand containing the AF adduct annealed to a 14mer, such that the 3'-hydroxyl primer terminus was four nucleotides before the modified guanine. Synthesis on this template by DNA polymerase I efficiently bypassed the AF adduct and produced full-length duplex 31mers. T7 DNA polymerase, on the other hand, was unable to utilize the AF-modified template though it was active on an identical unmodified one. The strand synthesized by DNA polymerase I was then separated from the modified strand, annealed to a complementary oligonucleotide, and the resulting heteroduplex cloned into M13. Each of the 49 clones isolated had sequences which indicated that cytidine had been incorporated opposite the AF-modified guanine.

Transcription by T7 RNA polymerase using benzo[a]pyrene-modified templates.

Synthesis of T7 RNA polymerase is inhibited by the presence of bulky adducts in the DNA template. Of the types of adducts tested, those formed by the potent carcinogen benzo[a]pyrene (B[a]P) caused the greatest inhibition. M13 DNA molecules containing a single late T7 RNA polymerase promoter have been prepared containing B[a]P adducts in either the displaced or template strand and these have been used as templates for in vitro RNA transcription by the T7 RNA polymerase. We find that the level of inhibition of RNA synthesis is substantially greater (greater than or equal to 10-fold) when adducts are positioned specifically in the template strand. Polyacrylamide gel analysis of the products synthesized off these strand-specifically modified templates showed that adducts situated in the template strand gave rise to discrete bands which presumably represent the termination of synthesis at the adduct site while the product derived from a template containing adducts in the displaced resembled that obtained using a native template.

Effects of benzo[a]pyrene-DNA adducts on a reconstituted replication system.

We have used a partially reconstituted replication system consisting of T7 DNA polymerase and T7 gene 4 protein to examine the effect of benzo[a]pyrene (B[a]P) adducts on DNA synthesis and gene 4 protein activities. The gene 4 protein is required for T7 DNA replication because of its ability to act as both a primase and helicase. We show here that total synthesis decreases as the level of adducts per molecule of DNA increases, suggesting that the B[a]P adducts are blocking an aspect of the replication process. Polyacrylamide gels indicate that a shorter DNA product is produced on modified templates and this is confirmed by determining the average chain lengths from the ratio of chain initiations to chain elongation. Gene 4 protein primed synthesis reactions display a greater sensitivity to the presence of B[a]P adducts than do oligonucleotide-primed reactions. By challenging synthesis on oligonucleotide-primed B[a]P-modified DNA with unmodified DNA, we present evidence that the T7 DNA polymerase freely dissociates after encountering an adduct. Prior studies [Brown, W. C., & Romano, L. J. (1989) J. Biol. Chem. 264, 6748-6754] have shown that the gene 4 protein alone does not dissociate from the template during translocation upon encountering an adduct. However, when gene 4 protein primed DNA synthesis is challenged, we observe an increase in synthesis but to lesser extent than observed on oligonucleotide-primed synthesis.(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.