mutational properties of the primary aflatoxin b1-dna adduct

Mutational Properties of the Primary Aflatoxin B1-DNA AdductAuthor(s): Elisabeth A. Bailey, Rajkumar S. Iyer, Michael P. Stone, Thomas M. Harris and JohnM. EssigmannSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 93, No. 4 (Feb. 20, 1996), pp. 1535-1539Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/38617 .

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Proc. Natl. Acad. Sci. USA Vol. 93, pp. 1535-1539, February 1996 Biochemistry

Mutational properties of the primary aflatoxin Bl-DNA adduct (abasic sites/SOS mutagenesis/chemical carcinogenesis)

ELISABETH A. BAILEY*, RAJKUMAR S. IYERtt, MICHAEL P. STONEt, THOMAS M. HARRISt, AND JOHN M. ESSIGMANN*?

*Department of Chemistry and Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA 02139; and tDepartment of Chemistry, Vanderbilt University, Nashville, TN 37235

Communicated by George H. Btichi, Massachusetts Institute of Technology, Cambridge, MA, November 6, 1995 (received for review September 18, 1995)

ABSTRACT The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7- Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G -* T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G -> T mutations of AFB1-N7-Gua, unlike those of the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern match- ing that in intact cells treated with the toxin. It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7- Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.

The fungal metabolite aflatoxin B1 (AFB1) contaminates the food supply in eastern Asia and sub-Saharan Africa, where it is associated with an increased incidence of hepatocellular carcinoma (HCC) (1). AFB1 requires metabolic conversion to its exo-8,9-epoxide (2, 3) in order to cause damage to DNA (4), the event that presumably initiates the genetic changes resulting in HCC (5). The AFB1 epoxide reacts with guanine to form a population of adducts (6), the principal of which both in vitro (3, 7-9) and in vivo (8, 10-12) is 8,9-dihydro-8-(N7-guanyl)- 9-hydroxyaflatoxin B1 (AFB1-N7-Gua). The positively charged imidazole ring of this adduct promotes depurination, resulting in apurinic (AP) site formation. Alternatively, under slightly basic conditions the imidazole ring of AFB1-N7-Gua opens to form the chemically and biologically stable AFB1 formami- dopyrimidine (AFB1-FAPY) (1). The initial AFB1-N7-Gua adduct, the AFB1-FAPY, and the AP site, individually or collectively, represent the likely chemical precursors to the genetic effects of AFB1.

The nature of mutations induced by electrophilic derivatives of AFB1 has been studied in several forward mutation assays. Upon examining AFB1 mutagenesis in the endogenous Es- cherichia coli lacI gene, Foster et al. (13) found that the predominant mutations induced by metabolically activated AFB, are GC --> TA transversions in cells induced for the SOS

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response and containing the mucAB mutagenesis-enhancing operon. Both GC -* TA and GC -- AT mutations are induced with equal efficiency in other systems by metabolically activated AFB1 (14) and AFB1 8,9-dichloride (15), a chemical model for the AFB1 epoxide. In human cells, replication of an AFB1-modified pS189 shuttle vector shows predominantly GC -> TA transversions (16, 17). In the c-Ki-ras oncogene of rat hepatocellular carcinomas, GC -- AT transitions in codon 12 are observed (18, 19). Recently, mutations in the p53 tumor suppressor gene of HCCs have been examined from humans residing where AFB1 exposure is a frequent occurrence. Approximately half of these HCCs contain GC -> TA transversions in the third position of codon 249 (AGG) of the p53 gene (20, 21). The same mutations are observed in human hepatocytes grown in culture (22).

The genetic studies cited above indicate that several mutations occur in DNA globally modified by AFB1, but the predominant mutation induced or selected in vivo appears to be the GC -> TA transversion. At least three DNA lesions- AFB1-N7-Gua, AFB1-FAPY, and the AP site-are candidates as the precursor(s) to the mutations induced by aflatoxin. It has been reasonably suggested that the premutagenic lesion responsible for the observed GC -- TA transversions may be the AFB1-induced AP site (13, 23, 24), since dAMP is the most common base inserted opposite AP sites in E. coli induced for the SOS response (25, 26). It is a formal possibility, however, that the parental adduct AFB1-N7-Gua could also give rise to this mutation. The present work was aimed at determining the relative contributions of the primary AFB1-N7-Gua adduct (see Fig. 1 Inset) and the AP site to the mutagenic spectrum of AFB1.

MATERIALS AND METHODS

Materials. Restriction enzymes and Sephadex G-50 Quick Spin columns were from Boehringer Mannheim. Bacterio- phage T4 polynucleotide kinase, T4 DNA ligase, and exonuclease III were from Pharmacia. Uracil DNA glycosylase was from GIBCO/BRL. 5-Bromo-4-chloro-3-indolyl 3-D-galactopy- ranoside (X-Gal) and isopropyl f3-D-thiogalactopyranoside (IPTG) were from Gold Biotechnology (St. Louis). [a-35S]dATP and Sequenase version 2.0 sequencing reagents were from Am- ersham. Plasmid preparation kits were from Qiagen (Chatsworth, CA). Bacteriophage M13mp7L2 genome was a gift of C. W. Lawrence (27). The cell lines used were DL7 (AB1157; lacAU169) (28), DL7/pGW16 (DL7; pGW16 mucAB) (28),

Abbreviations: AFB1, aflatoxin B1; AFB1-N7-Gua, 8,9-dihydro-8-(N7- guanyl)-9-hydroxyaflatoxin B1 adduct; AFB1-FAPY, aflatoxin B1 for- mamidopyrimidine adduct; ss, single stranded; RF, replicative form; AP, apurinic; RRF, restriction-resistant fraction; HCC, hepatocellular carcinoma. tPresent address: BioGenex, 4600 Norris Canyon Road, San Ramon, CA 94583.

?To whom reprint requests should be addressed.

1535



1536 Biochemistry: Bailey et al. Proc. Natl. Acad. Sci. USA 93 (1996)

GW5100 (JM103, P1-) from G. Walker (Massachusetts Insti- tute of Technology), and NR9050 (F' prolacIZAM15, Aprolac, suB) from R. Schaaper (National Institutes of Health). Oli- gonucleotide d(CCTCTTCGAACTC) was synthesized and purified, and a portion was used to prepare [AFB,-N7-Gua- d(CCTCTTCGAACTC)] (adduct at underlined base) as described (29). For details, see Bailey et al. (E.A.B., R.S.I., T.M.H., and J.M.E., unpublished).

Preparation of Singly Modified Genomes. Single-stranded (ss) M13 genomes containing a unique AFB1-N7-Gua adduct (M13-AFB1), AP site (M13-AP), or unmodified guanine were generated as described (E.A.B., R.S.I., T.M.H., and J.M.E., unpublished), except that unlabeled ATP (1 mM) was used for phosphorylation of the respective oligonucleotides (see Fig. 1). Briefly, a ss M13mp7L2 genome was linearized with EcoRI and annealed to a 53-mer uracil-containing scaffold oligonucleotide to generate a circular M13 genome containing a 13-base gap. The gap is complementary to a second oligonucleotide, d(CCTCTTCGAACTC), containing an Sfu I recognition sequence (underlined). The 5'-phosphorylated [AFB1- N7-Gua-d(CCTCTTCGAACTC)] oligonucleotide was ligated into the gapped genome, and the scaffold was removed by uracil DNA glycosylase and exonuclease III. A portion of the M13-AFB1 and the unmodified control genomes was heated at 80?C for 15 min at pH 6.6 to generate the AP site and the unmodified heated (unmodified A) genomes, respectively.

Transformation of E. coli Cells with Singly Modified Ge- nomes. Immediately after preparation, genomes were centri- fuged for 4 min at 2000 x g at 4?C through a Sephadex G-50 Quick Spin column preequilibrated in H20. The DNA was maintained on ice for no longer than 3 h, after which all genome transfections were complete. A portion (30 gl) of each reaction mixture was examined by gel electrophoresis and the presence of completely ligated genomes was confirmed by comigration with ss circular DNA.

E. coli DL7 and DL7/pGW16 cells were prepared for transformation by electroporation as described (30). Half of each culture was UV irradiated at 45 J/m2 (28), a fluence that proved optimal for induction of SOS-dependent mutagenesis (data not shown). For each transformation, 190 gl of the cell suspension was added to 5 gl of the DNA solution. The total amount of DNA in this volume was determined as described (E.A.B., R.S.I., T.M.H., and J.M.E., unpublished) and repre- sents -44 ng of unmodified genome, 36 ng of unmodified A genome, 24 ng of M13-AFB1 genome, and 17 ng of M13-AP genome. The mixture was transferred to a cold Bio-Rad gene pulser cuvette (0.2 cm) and electroporations were performed with a BTX electro cell manipulator 600 system set at 50 gF and 129 Ql. The electroporation field strength optimal for cell survival and transformation efficiency was 7.5 kV/cm for UV-irradiated (UV+) DL7 and DL7/pGW16 cells and 12.5 kV/cm for UV-unirradiated (UV-) DL7 and DL7/pGW16 cells. Immediately after electroporation, 1 ml of SOC medium (31) was added and a portion of the bacterial suspension was plated in the presence of NR9050 cells, X-Gal, and IPTG (32) to determine the transformation efficiency. Two milliliters more of SOC was added, and the mixture was incubated for 1.5-2 h at 37?C, after which the progeny phage were isolated from the supernatant.

Mutant Enrichment Procedure. E. coli DL7/pGW16 cells (UV+ and UV-) were transformed as described above with M13-AFB1, M13-AP, and control genomes. In each case progeny phage, representing -2 x 104 individual transformation events, were pooled from 8 to 16 independent transformations and used to produce replicative form (RF) DNA by using the Qiagen midiplasmid purification system. Mutations within the Shfu I restriction site, resulting from either AFB1- N7-Gua or the AP site, render the sequence refractory to digestion by Sifu I. Thus, the mutant population was enriched by digesting agarose gel-purified RF DNA (250 ng) with Sfu I

(30 units) at 37?C for 2 h in the buffer supplied with the enzyme. The reaction mixtures were diluted 1:100 in H20, and 100 gl (5 ng of DNA) was used to transform 100 gl of DL7 cells by electroporation as described above.

Mutation Frequency Determination. To compare directly the genetic effects of AFB1 and the AP site, the mutation frequency and specificity for each lesion was calculated in exactly the same manner. Therefore, mutation frequencies for both lesions in DL7/pGW16 cells represent the fraction of surviving genomes that results from the insertion of nucleo- tides dAMP, dGMP, and dTMP, but not dCMP, opposite the lesion. DNA used in the above transformations gave rise to colorless, dark-blue, and light-blue plaques. Dark-blue plaques resulted from restoration of the M13mp7L2 lacZ reading frame, which is out of frame by +2, upon ligation of the 13-mer oligonucleotide. Light-blue plaques originated from a G -> T transversion within the 5'-GAA-3' codon containing the AFB1-N7-Gua adduct, thus resulting in an in-frame ochre (5'-TAA-3'). Partial suppression of ochre codons, in E. coli NR9050 cells, yields light-blue plaques. Plaques resulting from all other adduct-induced base substitutions were dark blue. Colorless plaques resulted from large deletions in the lacZ gene as a result of genetic engineering proceduresS or were due to undigested parental M13mp7L2 DNA. Frameshift mutations within the target sequence would also yield colorless plaques.

RF DNA, prepared from DL7/pGW16 transformations, was treated (Sfu+) or not (Sfu-) with Sfu I, transformed into DL7 cells, and plated for plaque formation. The restriction- resistant fraction RRF (ratio of plaques from Sfu+ to those from Sfu-) was higher than the true mutant fraction of the lesion under study because the Sfu+ sample included some wild-type DNA that had evaded digestion and some large deletions attributable to genetic engineering procedures.? To calculate the true mutant fraction, a sampling of dark-blue and light-blue plaques was sequenced (33) from Sfu+ transformations. The fraction of each class that contained mutations targeted within the 6-base Sfu I site was determined. The mutation frequency (%) of the lesion was 102(RRF) (fraction of plaques in the RRF determined to be true mutants by sequencing).

Given that the predominant mutations observed for both AFB1-N7-Gua and the AP site in DL7/pGW16 cells were phenotypically selectable light-blue mutants (G -> T and AP

T, respectively), comparative mutation frequencies resulting from transformations of M13-AFB1 and M13-AP into DL7 cells (cells lacking pGW16) represent only the frequency of light-blue G -> T and AP -> T mutations, respectively, relative to the total number of light-blue plus dark-blue plaques. Sfu I mutant enrichment of the progeny phage from these transformations was carried out, however, to facilitate calculation of the frequency of light-blue plaques. Mutation frequencies represent an average of three ligations and subsequent transformations.

RESULTS

Mutation Frequencies of AFB1-N7-Gua and the AP Site. ss AFB1 and AP site-containing genomes (Fig. 1) were replicated within E. coli. Previous data suggested that AFB1-induced base substitution mutations in E. coli are dependent on the presence of mucAB mutagenesis-enhancing gene products and that the

ISequence analysis of the colorless plaques indicated that a significant proportion contained large deletions originating from exonuclease III digestion on the 3' side of the gapped genome and subsequent ligation across the gap; these genetic engineering mutants lacked the Sfu I recognition sequence and were not selected against during the mutant enrichment process. A small percentage of these deletions resulted in restoration of the lacZ frame resulting in dark-blue plaque formation.



Biochemistry: Bailey et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1537

Eco RI Site 3'-U-UU-U-UU-5'

Uracil N | M13mP7L2 1- < S n Containing

Eco RI 53-mer Scaffold

3'-U-UU-U-UU-5' AFB1-N7 Ligase

zN7-Gi~1 T '- pCCTCTTCGAACTC-3' AF1N-ua I _

Sfu I Recognition Sequence C0H

Uracil DNA Glycosylase, Exonuclease III o 0H

0 OH AFB1-N7-Gua AP Site R X

~~Z ~~W H2N N N7-Gua 15mm ~~~~~~~dR m ~~~15 min t ,3 ' 7G

FIG. 1. Construction of singly modified ss genomes containing AFB1-N7-Gua (Inset) or the AP site. See text for details.

normal E. coli analog, umuDC, is not sufficient (23). Thus, we determined the mutation frequency of AFBI-N7-Gua in E. coli cells (DL7) containing pGW16, a plasmid that carries a mucAB allele that shows enhanced SOS mutagenic processing compared to wild-type MucAB (34, 35). Mutation frequencies were determined in both UV- and UV+ cells in order to examine the effect of the normal E. coli SOS mutagenic processing analog umuDC.

The mutation frequency of AFB1-N7-Gua in UV-irradiated DL7/pGW16 cells was 4.0% (Fig. 2A, bar 6), including 2.9% G -- T (bar 5). The mutation frequency of AFBI-N7-Gua in UV-unirradiated DL7/pGW16 cells was 2.9% (bar 4) including 2. 1% G -> T (bar 3). The frequencies above were calculated from pooled phage and were confirmed by the analysis of two individual ligations and subsequent transformations. These steps guaranteed that the average mutation frequency and specificity of pooled phage reflected that of an individual ligation/transformation event.

The mutational data on AFB,-N7-Gua were similar when UV+ and UV- DL7/pGW16 cells were used. To test whether DL7/pGW16 cells were expressing the mucAB SOS mutagenesis-enhancing gene products even without induction by UV

5 ~50?

ELXL

n U - + - + UV- + - + * I DL7 II DL7/pGW16 I DL7 LDL7/PGW18

1 2 3 4 5 6. 7 8 9 10 1112 AFB1-N7-Gua AP Site

FIG. 2. Mutation frequencies of AFB1-N7-Gua (A) and the AP site (B). Open bars, % of all progeny that was mutant; shaded bars, % of all progeny that was G -~T (A) or AP -~T (B). In DL7 cells, only G - > T or AP -*T percentages were determined; for each modified genome, 24 light-blue plaques (obtained from Sfu I enrichment) were sequenced. Controls for this experiment gave the following results. For the unmodified genome in DL7/pGW16 cells, mutation frequencies were 0.06% (UV-) and 0.20% (UV+); in DL7 cells, the values were 0.04% (UV-) and 0.06% (UV+). Mutation frequencies of the unmodified / control genome in DL7/pGW16 cells were 0.07% (UV-) and 0.19% (UV+) and 0.04% (UV-) and 0.05% (UV+) in DL7 cells.

light, and also to compare the specificity of AFB,-induced to AP site-induced mutations (discussed below), we examined in parallel the mutation frequency and specificity of an AP site, a known SOS-dependent mutagen (25, 26). The data in Fig. 2B show that the total mutation frequency of the AP site control was not very different in UV- and UV+ DL7/pGW16 cells: 31% (bar 10) including 16% AP -> T (bar 9) (UV-) and 50% (bar 12) including 37% AP -- T (bar 11) (UV+). By contrast, the mutation frequencies of the AP site in UV- and UV+ DL7 cells (i.e., cells lacking mucAB) were 0.6% (bar 7) and 18% (bar 8) AP -> T, respectively. The G -> T mutation frequencies of AFB,-N7-Gua (Fig. 2A) in UV- and UV+ DL7 cells were <0.05% (bar 1) and 0.5% (bar 2), respectively. These data indicate that, in contrast to DL7/pGW16 cells, UV induction of SOS functions in DL7 cells markedly enhances (10- to 30-fold) AFB,-N7-Gua and AP site mutagenesis. The evident expression of the SOS mutagenic processing phenotype in DL7/pGW16 cells, without preinduction with UV, is possibly due to a point mutation in pGW16 within the LexA repressor-binding site of the mucAB operator that weakens LexA protein binding to the operator (35). This results in an elevated level of mucAB expression (35), seemingly sufficient to effect an SOS processing phenotype in these unirradiated cells. The feasibility of this model is evidenced by recent studies (36).

Mutational Specificity of AFB,-N7-Gua and the AP Site. The mutational specificity of AFB1-N7-Gua and the AP site were examined in both UV- and UV+ DL7/pGW16 cells (Fig. 3). The mutations observed for AFB1 were predominantly G -> T transversions targeted to the original site of the adduct (base 6240) in both UV- cells (74%) and UV+ cells (73%). G -- A transitions occurred at frequencies of 18% in UV- cells and 13% in UV+ cells. G -> C transversions were infrequent at 1.4% in UV- cells and 2.5% in UV+ cells.

The most striking of all the mutations observed were base substitutions at the base 5' to the modified guanine (base 6239): C -> T (7%) in UV- cells, and C -- T (10%) and C > A (2.5%) in UV+ cells. Evidence that these nontargeted 5' mutations were due to the aflatoxin moiety was obtained by examining the mutational specificity of the AP site. As indicated

UV Treatment AFB,-N7-Gua AP Site of Cells #/Total % Dist. #/Total % Dist.

T ---(15/59) ---7. 0 -- 0 -- c ----- (3/59) --- 1.4 - -(25/85) - - 14.5 -- A ---- (36/59) - - 17.6 -- -(59/85) -- 34.0 - - T ---- (48/48) - - 74.0 - - (24/24) - - 51.5 - -

5'-TTCG*AA-3'

T------ (35/96) -10.0- ------- 0 - - A F- - ----- (7/96) - -0- 2.5 ------------- 0-

+ 0 ---- (11/96) -- - 2.5 --- - (17/80)--- 6.0-- A ---- (40/96) - - 12.5 -- - (61/80) - - 20.0 - - T ---- (96/96)--72.5 -- - (24/24)-- 74.0__

FIG. 3. Type and percentage distribution of AFB1-N7-Gua and AP site-induced mutations in E. coli DL7/pGW16. G*, position of lesion. A total of 926 dark-blue sequences was examined from M13-AFB1 and M13-AP transformations collectively. The number of dark-blue mutants sequenced with mutations in the Sfu I restriction site was 96 for AFB1-N7-Gua (UV+), 59 for AFB1-N7-Gua (UV-), 80 for AP site (UV+), and 85 for AP site (UV-). The number of light-blue mutants sequenced (G -- T or AP -> T) was 96 for AFB1-N7-Gua (UV+), 48 for AFB,-N7-Gua (UV-), 24 for AP site (UV-), and 24 for AP site (UV+). The number sequenced for each individual mutation is shown in parentheses and is represented as the number sequenced/total number of dark-blue or light-blue true mutants sequenced (#/Total). Mutants at <0.5% of the total number of mutants are not reported.



1538 Biochemistry: Bailey et al. Proc. Natl. Acad. Sci. USA 93 (1996)

in Fig. 3, the predominant mutations observed were AP -> T (52% in UV- cells and 74% in UV+ cells), followed by AP -> A (34% in UV- cells and 20% in UV+ cells) and AP -- C (14.5% in UV- cells and 6% in UV+ cells). Only two tandem mutations (not shown in Fig. 3) involving base 6239 were observed for the AP site in UV+ cells: Cp(AP) --ApC (0.4%) and Cp(AP) -- TpC (0.4%). One tandem mutation was observed in UV- cells: Cp(AP) -> TpC (0.6%). Thus, unlike the results obtained with AFB1-N7-Gua, no significant number of mutations was observed at base 6239, indicating that the mutations induced at base 6239 are a signature mutation of the aflatoxin moiety and not due to an AP site.

Earlier work has shown that frameshift mutations occur in DNA modified globally by reactive derivatives of AFB1 (15, 37). It was therefore of interest to determine to what extent these may have been caused by AFB1-N7-Gua in our system. This analysis was complicated, however, owing to a back- ground of "genetic engineering" mutations;$ these mutations had the same phenotype (colorless) as the putative frameshifts. In an attempt to determine the level of AFB1-induced frameshift mutations, a sufficient number of colorless plaques was sequenced so that we are able to set an upper limit of their occurrence at 0.2%, thus indicating that at least in our system AFB,-N7-Gua-induced frameshifts are very infrequent events.

DISCUSSION DNA globally modified with activated forms of AFB1 contains at least three DNA lesions: AFB,-N7-Gua, AFB1-FAPY, and the AP site. The goal of our work was to determine the relative contributions of AFB1-N7-Gua and the AP site to the mutagenic spectrum of AFB1. The predominant mutation of AFB1- N7-Gua (Fig. 3) was the G -- T transversion (-75% of all mutations), similar to that observed previously with metabolically activated AFB1 in E. coli induced for the SOS response and containing the mucAB mutagenesis-enhancing gene products (13) and in mammalian systems (16, 22). A possible premutagenic lesion responsible for this mutation is the AP site that results from the facile depurination of AFB1-N7-Gua. We have considered the possibility that an AFB,-N7-Gua- derived AP site could have been responsible for the observed mutations. It is unlikely that the M13-AFB1 genomes used for our studies were contaminated with a small amount of M13-AP genome generated during genome construction procedures. Genome characterization data (E.A.B., R.S.I., T.M.H., and J.M.E., unpublished) suggest that an enzymatic step (exonuclease III treatment) applied at the final stage of genome construction efficiently inactivated any contaminating AP site genome, leaving M13-AFB1 2 95% pure with a 5% contamination of unmodified genome. It was more likely that the AP site could be responsible for the observed mutations through spontaneous or enzymatic in vivo depurination of AFB,-N7-Gua prior to DNA replication. AFB1-N7-Gua, however, is removed from rat liver DNA with a half-life of 7.5 h (11), suggesting that the adduct was intact during the short time span of replication of M13-AFB1 in E. coli (32). Although we predict, based on the aforementioned considerations, that the AP site does not effect the observed G -- T transversions, we could not on the above grounds rule out depurination of AFBI-N7-Gua as the event leading to these mutations. To address this issue more definitely, we compared the mutational specificity of AFB,-N7-Gua and the AP site and, in addition, the genetic backgrounds required to produce the observed mutations.

The mutational specificity of the AP site (Fig. 3) is similar to what others have observed in E. coli induced for the SOS response (25, 26). Thus, the predominant mutation caused by both AFB1 and the AP site appears to result from insertion of dAMP opposite the site of the adduct. Although the principal mutations of both the AP site and AFB1-N7-Gua required

some form of SOS mutagenic processing, the relative contributions of UmuDC and MucAB as effectors of these mutations differed. The data in Fig. 2 represent three SOS mutagenic environments: largely umuDC-mediated mutagenesis (UV+ DL7), largely mucAlB-mediated mutagenesis (UV- DL7/ pGW16), and combined umuDC- and mucAB-mediated mutagenesis (UV+ DL7/pGW16). The data suggest that the umuDC gene products enable the insertion of dAMP opposite the AP site (AP -* T) at a frequency similar to that resulting from the mucAB gene products alone (18% and 16%; Fig. 2B, bars 8 and 9, respectively). Furthermore, the combination of umuDC and mucAB expression in UV+ DL7/pGW16 cells doubles the AP -> T mutation frequency (bar 11), indicating that both UmuDC and MucAB contribute significantly to the frequency of AP -* T mutations. However, in contrast to AP site mutagenesis, AFB1-N7-Gua mutagenesis relies much more heavily on the gene products of mucAB than umuDC to effect G -- T transversions; the umuDC and mucAB gene products induce G -* T transversions at frequencies of 0.5% vs. 2.1% (Fig. 2A, bars 2 and 3, respectively). In addition, and in contrast to AP site mutagenesis, the combined expression of umuDC and mucAB (UV+ DL7/pGW16) (bar 5) resulted in only a small increase in AFB1-induced G - T transversions

A 5' 39

~~~~~~~~~5 3 %

GC yTA

TA /6240

B

I~ ~ ~ ~A sCtoT9

73 '~~~~~~~~3

FIG. 4. (A) Potential energy minimized structure of the central portion of [AFBi-N7-Gua-d(CCTCTTCGAACTC) d(GAGTTCGA- AGAGG)]. Model was derived from and is consistent with NMR data for [AFBi-N7-Gua-d(ATCQAT) d(ATCGAT)], where the AFB1 moiety is intercalated on the 5' face of the modified guanine (40). (B) Model based on the structure mnA depicting the AFB1-N7-Gua adduct at the moment of replication. It is proposed that the AFB1 moiety shifts in the 5' direction, perhaps as a consequence of normalization of the C-G pair at position 6240. Q6239 may rotate out of the helix to accommodate the AFB1 moiety. The C -CT transition at position 6239 occurs because of the insertion of adenine across from the AFB1 moiety.



Biochemistry: Bailey et al. Proc. Natl. Acad. Sci. USA 93 (1996) 1539

(from 2.1% to 2.9%). The AFBI-N7-Gua data are consistent with studies carried out on DNA globally modified with AFBI showing a strong MucAB dependence for mutagenesis (23). The mechanisms underlying the different biochemical activi- ties of UmuDC and MucAB on AFBI and AP site mutagenesis are unclear. From the standpoint of our study, however, it is important that we were able to take advantage of this difference to reveal that the AFBI -induced G -> T transversions are promoted through mechanisms not involving in a predominant manner the simple depurination of the AFBI-N7-Gua adduct.

The most striking difference between the mutational specificity of AFBI-N7-Gua and the AP site is represented by a significant proportion (1.3%) of mutations targeted to M13 base 6239 (5' to the AFBI modified guanine) (Fig. 3). Our data indicate that these mutations are not induced by the AP site. Given that the total mutation frequency for AFBI-N7-Gua in our system is high (4%) compared to many helix-distorting DNA adducts (30, 38, 39), 13% of the mutations represent a significant mutant population at 0.5%.

The observed mutational asymmetry disposed to the 5' side of the adduct is in accord with the molecular architecture of AFBI-N7-Gua established by NMR (40) showing the aflatoxin moiety of the adduct intercalated on the 5' face of guanine (Fig. 4). It is unknown to what extent the intercalative state exists at the moment of replication, but it is reasonable to speculate that the aflatoxin moiety remains proximal to the 5' base during DNA synthesis, thus potentially interfering with 5' base pairing. The observation that mutations occur at base 6239 suggests that mutational spectra obtained in previous studies from DNA globally modified with AFBI may harbor mutations at bases 5' to potentially modified guanines. Since guanine residues that are flanked by A+T-rich sequences are poor targets for modification by AFB, (41, 42), it is conceivable that the majority of 5' mutations would reside in 5' cytosines or guanines; these would mistakenly have been scored as resulting from either an additional AFB,-modified guanine in the same strand (GGX) or an AFBI-modified guanine in the opposite strand (CGX) (underlined base is potentially modified base; italicized base is potentially mutated 5' base). It is noteworthy that only through the use of a specifically placed AFBI-N7-Gua adduct were we able to present a mechanism for AFBI mutagenesis that involves not only the modified base but also the base 5' to the intercalated aflatoxin moiety (a cytosine in this study). It will be of interest to examine how bases other than cytosine 5' to the adduct influence this mechanism for semitargeted mutagenesis.

This work strongly suggests that the aflatoxin moiety of the primary aflatoxin adduct, AFBI-N7-Gua, gives rise to a significant proportion of the observed AFBI-induced mutations. Our data indicate that the aflatoxin moiety gives rise to 5' nontargeted mutations at base 6239 and to targeted G -> T transversions at base 6240. It will be of interest to examine the mutagenic potential of the more chemically stable AFB1- induced adduct AFB,-FAPY in comparison to the mutational spectrum obtained in this study for AFBI-N7-Gua and the AP site.

We thank C. Lawrence and R. Schaaper for strains, E. Escue for molecular modeling, and A. Barrasso and M. Smela for technical assistance. K. Yarema and D. Treiber are thanked for helpful discus- sions. This study was supported by Grants CA52127, CA55678, and E037755 from the National Institutes of Health.

1. Busby, W. F., Jr., & Wogan, G. N. (1984) in Chemical Carcino- gens, ed. Searle, C. (Am. Chem. Soc., Washington, DC), pp. 945-1136.

2. Swenson, D. H., Lin, J.-K., Miller, E. C. & Miller, J. A. (1977) Cancer Res. 37, 172-181.

3. Essigmann, J. M., Croy, R. G., Nadzan, A. M., Busby, W. F., Jr., Reinhold, V. N., Buchi, G. & Wogan, G. N. (1977) Proc. Natl. Acad. Sci. USA 74, 1870-1874.

4. Miller, E. C. (1978) Cancer Res. 38, 1479-1496. 5. Harris, C. C. (1991) Cancer Res. 51, 5023s-5044s. 6. Essigmann, J. M., Green, C. L., Croy, R. G., Fowler, K. W.,

Buchi, G. H. & Wogan, G. N. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 327-337.

7. Martin, C. N. & Garner, R. C. (1977) Nature (London) 267, 863-865.

8. Lin, J.-K., Miller, J. A. & Miller, E. C. (1977) Cancer Res. 37, 4430-4438.

9. Groopman, J. D., Croy, R. G. & Wogan, G. N. (1981) Proc. Natl. Acad. Sci. USA 78, 5445-5449.

10. Croy, R. G., Essigmann, J. M., Reinhold, V. N. & Wogan, G. N. (1978) Proc. Natl. Acad. Sci. USA 75, 1745-1749.

11. Croy, R. G. & Wogan, G. N. (1981) Cancer Res. 41, 197-203. 12. Croy, R. G. & Wogan, G. N. (1981) J. Natl. Cancer Inst. 66,

761-768. 13. Foster, P. L., Eisenstadt, E. & Miller, J. H. (1983) Proc. Natl.

Acad. Sci. USA 80, 2695-2698. 14. Sahasrabudhe, S., Sambamurti, K. & Humayun, M. Z. (1989)

Mol. Gen. Genet. 217, 20-25. 15. Sambamurti, K., Callahan, J., Luo, X., Perkins, C. P., Jacobsen,

J. S. & Humayun, M. Z. (1988) Genetics 120, 863-873. 16. Trottier, Y., Waithe, W. I. & Anderson, A. (1992) Mol. Carcinog.

6, 140-147. 17. Levy, D. D., Groopman, J. D., Lim, S. E., Seidman, M. M. &

Kraemer, K. H. (1992) Cancer Res. 52, 5668-5673. 18. Soman, N. R. & Wogan, G. N. (1993) Proc. Natl. Acad. Sci. USA

90, 2045-2049. 19. McMahon, G., Davis, E. F., Huber, L. J., Kim, Y. & Wogan,

G. N. (1990) Proc. Natl. Acad. Sci. USA 87, 1104-1108. 20. Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J. &

Harris, C. C. (1991) Nature (London) 350, 427-428. 21. Bressac, B., Kew, M., Wands, J. & Ozturk, M. (1991) Nature

(London) 350, 429-431. 22. Aguilar, F., Hussain, S. P. & Cerutti, P. (1993) Proc. Natl. Acad.

Sci. USA 90, 8586-8590. 23. Foster, P. L., Groopman, J. D. & Eisenstadt, E. (1988) J. Bacte-

riol. 170, 3415-3420. 24. Kaden, D. A., Call, K. M., Leong, P. M., Komives, E. A. & Thilly,

W. G. (1987) Cancer Res. 47, 1993-2001. 25. Loeb, L. A. & Preston, B. D. (1986) Annu. Rev. Genet. 20,

201-230. 26. Lawrence, C. W., Borden, A., Banerjee, S. K. & LeClerc, J. E.

(1990) Nucleic Acids Res. 18, 2153-2157. 27. Banerjee, S. K., Borden, A., Christensen, R. B., LeClerc, J. E. &

Lawrence, C. W. (1990) J. Bacteriol. 172, 2105-2112. 28. Lasko, D. D., Harvey, S. C., Malaikal, S. B., Kadlubar, F. F. &

Essigmann, J. M. (1988) J. Biol. Chem. 263, 15429-15435. 29. Gopalakrishnan, S., Stone, M. P. & Harris, T. M. (1989) J. Am.

Chem. Soc. 111, 7232-7239. 30. Wood, M. L., Dizdaroglu, M., Gajewski, E. & Essigmann, J. M.

(1990) Biochemistiy 29, 7024-7032. 31. Hanahan, D. (1985) DNA Cloning: A Practical Approach (IRL,

Oxford), pp. 109-135. 32. Messing, J. (1983) Methods Enzymol. 101, 20-78. 33. Meeker, A. K., Li, Y.-K., Shortle, D. & Stites, W. E. (1993)

BioTechniques 15, 370-372. 34. Walker, G. C. (1978) J. Bacteriol. 133, 1203-1211. 35. McNally, K. P., Freitag, N. E. & Walker, G. C. (1990)J. Bacteriol.

172, 6223-6231. 36. Hauser, J., Levine, A. S., Ennis, D. G., Chumakov, K. M. &

Woodgate, R. (1992) J. Bacteriol. 174, 6844-6851. 37. Refolo, L. M., Bennett, C. B. & Humayun, M. Z. (1987) J. Mol.

Biol. 193, 609-636. 38. MacKay, W., Benasutti, M., Drouin, E. & Loechler, E. L. (1992)

Carcinogenesis 13, 1415-1425. 39. Basu, A. K., Wood, M. L., Niedernhofer, L. J., Ramos, L. A. &

Essigmann, J. M. (1993) Biochemistry 32, 12793-12801. 40. Gopalakrishnan, S., Harris, T. M. & Stone, M. P. (1990) Bio-

chemistry 29, 10438-10448. 41. Muench, K. F., Misra, R. P. & Humayun, M. Z. (1983) Proc. Natl.

Acad. Sci. USA 80, 6-10. 42. Benasutti, M., Ejadi, S., Whitlow, M. D. & Loechler, E. L. (1988)

Biochemistry 27, 472-481.



mutational properties of the primary aflatoxin b1-dna adduct

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