Proc. Natl. Acad. Sci. USAVol. 81, pp. 6271-6275, October 1984Biochemistry

In vivo mutagenesis by 06-methylguanine built into a unique site ina viral geno'me

(chemical carcinogenesis/O6-alkylguanine/carcinogen-DNA adducts)

EDWARD L. LOECHLER, CALVERT L. GREEN*, AND JOHN M. ESSIGMANNtLaboratory of Toxicology, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, MA 02139

Communicated by James A. Miller, June 11, 1984

ABSTRACT The mutagenicity of 06-methylguanine(O6MeGua), a chemical carcinogen-DNA adduct, has- beenstudied in vivo by using a single-stranded M13mp8 genome inwhich a single OMeGua residue was positioned in the uniquerecognition site for the restriction' endonuclease Pst I. Trans-formation of Escherichia coli MM294A cells with this vectorgave progeny phage, of which 0.4% were mutated in their Pst Isite. In a separate experiment, cellular levels of O'MeGua-DNA methyltransferase (an' O6MeGua-repair protein) weredepleted by treatment with N-methyi-N'-nitro-N-nitrosoguani-dine (MNNG) prior to viral DNA uptake. In these cells, themutation frequency due to 06MeGua increased with increas-ing MNNG dose (the highest mutation frequency observed was20%). DNA sequence analysis of 60 mutant genomes revealedthat 06MeGua induced exclusively G-to-A transitions.

An important step in chemical carcinogenesis is thought tobe the formation of covalent abducts between DNA and re-active forms of chemical carcinogens (1). Misreplication ormisrepair of these lesions could cause mutations, which inprinciple could initiate malignant transformation of somaticcells (2, 3). Because carcinogens react with DNA to producea structurally diverse population of adducts, it is difficult todetermine the contribution of each DNA lesion to theamount and type of induced mutation. The approach wehave taken for establishing the relationship between adductstructure and biological effects is to situate individual ad-ducts at defined sites in genomes, allow enzymatic process-ing to occur in vivo, and then assess the resulting geneticchange(s) both qualitatively and quantitatively.

In this work, we have examined the mutagenic activity of06-methylguanine (O6MeGua), a DNA adduct formed bycertain alkylating agents (4). This lesion has been the focusof much recent attention because alkylating agent-inducedmutagenesis in cells and carcinogenesis in animals are oftencorrelated with its presence or persistence in DNA (5-10).Previously, we described the construction of an M13mp8genome containing an O6MeGua residue at a unique site(O6MeGua-Ml3mp8) (11). Using this vector, we report herethat O6MeGua, in isolation from all other lesions, produces apattern of in vivo mutagepesis similar to that found with al-kylating agents.

MATERIALS AND METHODSExonuclease III was obtained from New England Biolabs.Escherichia coli JM103 and MM294A were obtained from U.RajBhandary and K. Backman, respectively. JM103 cells(12) are F', which allows M13mp8 infection, and lacZ-.MM294A cells are F- and ada' [i.e., wild type (wt) for theinduction of O6MeGua-DNA methyltransferase (O6MeGuaMeTase)]. All other apparatus, media, and enzymes were asdescribed (11).

Isolation of Pst I-Insensitive Mutants Derived from O6Me-Gua-Ml3mpp8. In previous work, an M13mp8-gapped duplexgenome was constructed missing the central four bases (T-G-C-A) from one strand of the Pst I site (11). An O6MeGua-containing tetranucleotide (Tpr6GpCpA) was ligated intothe four-base gap, giving double-stranded (ds) O6MeGua-M13mp8. The nine-step method used to isolate Pst I-insensi-tive Mutants from single-stranded (ss) O6MeGua-Ml3mp8 isdiagrammed in Fig. 1 and described below. As a control, 100ng of unmethylated M13mp8 DNA [viral (+) strand] was tak-en through the same procedure.

Step 1; Transformation by O6MeGua-MJ3mp8 and isola-tion of progeny phage. ds 06MeGua-M13mp8 (200 ng persample) was denatured in 0.1 M NaOH for 1 min, neutralizedwith HCl, and adjusted to 30 mM CCl2/100 mM NaCI/0.75mM EDTA/10 mM Tris HCl, pH 8.0, containing bovine se-rum albumin at 1.5 mg/ml (final volume, 0.1 ml). The result-ant ss DNA was mixed with =2 x 109 transformation-com-petent E. coli MM294A cells (30 mM CaCl2/10 mMTris HCl, pH 8.0; final volume, 0.2 ml) and left on ice for 60min; DNA renaturation was shown not to occur under theseconditions. Subsequently MNNG was added to final con-centrations ranging from 0 to 50 Mg/ml (doses are listed inTable 1), and 2 min later the samples were heat-shocked(420C) for 1.5 min. Finally, samples were chilled on ice for1.5 min, 2 ml ofLB medium (prewarmed to 37iC) was added,and cells were incubated for 2 hr at 370C. The cells werepelleted, leaving phage in the supernatant.

Step 2; Isolation ofRF DNA from progeny phage. Phage(1 x 106) were preadsorbed to 5 x 1 JM103 cells for 15 minand then added to 10 ml of 2 x YT medium and grown over-night. RF I DNA was isolated as described (12).

Steps 3-6; Enrichment of mutant phage RF resistant toPst I cleavage. RF DNA (=1 lig) was treated with Pst I (20units); this treatment linearized wt molecules but left mu-tants intact. Treatment with exonuclease III (25 units for 20min at 370C, followed by 10 min at 650C) degraded the linearmolecules, as monitored by agarose gel electrophoresis. Themutant-enriched RF population was retransformed intoMM294A cells (step 4). In step 5, the progeny phage wereused to infect JM103 cells to produce a second RF popula-tion. We found that 1-2% of wt M13mp8 RF DNA remainedafter the Pst I/exonuclease III treatment (data not shown).Thus, to ensure that all RF molecules capable of producingwt phage had been eliminated, the cycle of Pst I cleavage,

Abbreviations: 06MeGua, 06-methylguanine; 06MeGua-M13mp8,genome of M13mp8 in which the first guanine of the Pst I site wasreplaced with O6MeGua; ss, single-stranded; ds, double-stranded;MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; O6MeGua Me-Tase, O6MeGua-DNA methyltransferase; RF, replicative form;IPTG, isopropyl P-D-thiogalactopyranoside; X-Gal, 5-bromo-4-chloro-3-indoyl f-D-galactopyranoside; T1, T2 and T3, first, secondand third transformations, respectively; MF, mutation frequency;wt, wild type.*Present address: AMGen, Inc., Thousand Oaks, CA 91320-1789.tTo whom reprint requests should be addressed.

6271

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6272 Biochemistry: Loechler et al.

Double- StrandedO6MeGua-M13mp8

06MeCTGCAGGAC GTC- (+)

(- iI

NaOH

O6MeG A C G TC-C TG CAG-' H

(+)-

Single- StrandedO6MeGua- Ml 3mp8

oMe.CT CAG-

v6255 (+)

06meGACGTC

6256w (-)i~~~~~~~~~~~~~~~~~~~~

TransformPhage Infect R F 1) Ps)(MM294A) P M103) DNA 2)Exz

t ~ ~~~~~RepeatStops Ph Infect Individual(a and (JMI03) PlaquesM

,-I Transform- Pha Infect RF-~~~-~~~~Phge -

o,& (MM294A) (JM103) DNA

Infect Single- ( Sequence- Stranded Anlyi(MO) DNA

FIG. 1. Isolation of O6MeGua-derived mutants. Encircled numbers denote procedural steps as described in Materials and Methods; T1, T2,and T3 are the three. E, coli transformation steps; 6255 and 6256 are the M13mp8 genome locations of O6MeGua in the (+) and (-) strands,respectively.

exonuclease III treatment, and transformation was repeated(step 6).

Steps 7-9; DNA sequence characterization ofmutants. Todetermine DNA sequence changes induced by the adduct,phage generated after step 6 were plated on JM103 cells toproduce individual p14pues. ss phage DNA was isolated fromsingle plaques (12) anrd sequenced (13).Mutation Frequency (MF) Determination. MFs were deter-

mined by three methods. 'The principal method involvedplating the phage produced after both the first and thirdtransformations (T1 and T3, respectively; Fig. 1) on JM103cells on M13 minimal agar, on which all phage (mutant aswell as wt) produced white plaques. Individual plaques werepicked randomly with toothpicks and stabbed onto platesseeded with JM103 cells in the presence of isopropyl -D-thiogalactopyranoside (IPTG) and 5-bromo-4-chloro-3-in-doyl f3-D-galactppyranoside (X-Gal). Upon incubation, threeplaque colors were observed (dark blue, light blue, andwhite), which were used to calculate MFs as described inResults. The second method of MF determination entailedplating the phage mixture produced aftf jl1 on JM103 cellsin the presence'bf IPTG and X-Gal and then randomly select-ing indivicpal dark-blue plaques for DNA sequencing (13).The frequency of mutant and wt sequences was calculatedon the basis of the sequencing results. The third method in-volved applying the "adenine" DNA sequencing reaction(13) to ss DNA isolated from the total phage populatiqn pro'duced after T1 of Fig. 1; the details are given in Fig. 3.

RESULTSTo investigate mutagenesis by O6MeGua in vivo, a dsM13mp8 DNA molecule was constructed in which a guanineresidue was replaced by O6MeGua in the unique recognitionsite for Pst I (figure LA of ref. 11). This vector, 06MeGua-M13mp8, had a uniquely positioned nick in the DNA strandopposite that containing O6Me(3ua; thus, alkali denaturationyielded ss circular adduct-containing viral DNA molecules(Fig. 1). Genomes containing O6MeGua at positions 6255and 6256 in the (+) and (-) strands, respectively, were pro-duced in equal amounts, and the mixture was used to trans-form'E. coli MM294A cells (Fig. 1, T1). [Alkali treatmentensured that DNA strands with gaps or nicks would not yieldphage because ss linear DNA does not transform efficiently(14).] In the MM294A cells, viral DNA replication occurred,and a mixture of wt and mutant phage was'produced. Subse-quently, these phage were used to infect E. coli JM103 cells,and replicative form (RF) DNA was isolated. RF DNA withmutations in the six-base recognition site for Pst I was insen-sitive to cleavage by Pst I, and this property was used toeliminate wt material and, thus, to isolate mutants.

Genetic Analysis: Characterization of Mutants. After themutant isolation procedure was completed (i.e., after T3 ofFig! 1) and individual plaques were plated in the presence ofIPTG and X-Gal, three plaque colors were observed: darkblue, light blue, and white. These mutant species were char-acterized by DNA sequencing.One mutant class derived from Pst I-insensitive DNA pro-

duced dark-blue plaques on IPTG/X-Gal plates. Of 14 dark-blue mutants sequenced, 10 showed a G-to-A transition atposition 6255 of the M13mp8 genome (Fig. 2). Presumably,O6MeGua located at this position in the (+) strand was mis-replicated in vivo to produce this single base change. Themutation occurred in the lacZ' fragment clpned into M13mp8(12); this fragment complements a deletion at the 5' end ofthe JM103 chromosomal lacZ gene. The G-to-A change atposition 6255 did not affect complementation; thus, phagewith this mutation produced dark-blue plaques in the pres-

(-) StrandM u tant

Wi IdType

(+) StrandM u t a n t

ACGT

GA C G T

_bA C GT AC G T

FIG. 2. DNA sequences of wt M13mp8 and of mutants derivedfrom O6MeGua-Ml3mp8. A primer complementary to the M13mp8sequence from base pairs 6284 to 6298 (Bethesda Research Labora-tories) was annealed to the viral (+) strands. The chain terminationprocedure of DNA sequencing was used (13), followed by electro-phoresis through a 12% polyacrylamide gel. This autoradiogram is ofthe portion of the gel that contains the Pst I site of wt, (+)-strandmutant, and (-)-strand mutant phage genomes; mutant DNAs wereisolated from phage produced after T3 of Fig. 1. The only sequencechange in the (-)-strand mutant, isolated from phage producinglight-blue plaques, was a G-to-A transition at position 6256. Theonly difference in the (+)-strand mutant, isolate; from phage pro-ducing dark-blue plaques, was a C-to-T transition at position 6255,which indicates that in the complementary (+) strand, a G-to-Achange had occurred. A total of 38, 22, and 34 mutants arising fromdark-blue, light-blue, and white plaques, respectively, were se-quenced; all showed the expected base changes.

Proc. NatL Acad Sci. USA 81 (1984)

Proc. Natl. Acad. Sci. USA 81 (1984) 6273

ence of IPTG and X-Gal. For the purpose of MF calculations(presented below), these mutants, which gave rise to dark-blue plaques and base-pair substitutions originating in the(+) strand, are designated M~bThe remaining four (of 14) mutants in the dark-blue class

were revealed by DNA sequencing to be deletion mutationsthat had lost the Pst I site but had maintained the correctreading frame of the lacZ' fragment. The origin of these mu-

tants was not investigated, but their size and location (-30base pairs, extending 5' to 3' from the border of the Pst Isite) suggests that they arose during the construction ofO6MeGua-M13mp8 (11). An imperfect T4 polymerase reac-

tion may have created duplex genomes with =30-base gaps,and by a mechanism recently proposed (15), DNA ligasecould have closed these gaps in vitro, resulting in the ob-served deletions. These mutants do not appear to have beenO6MeGua-derived because their frequency did not increaseunder treatments described below that enhanced the yield ofO6MeGua mutants. This was a minor mutant class, repre-senting 0.1% of the phage produced at T1.A second class of O6MeGua-induced mutant phage gave

light blue plaques when plated on IPTG/X-Gal medium. TheDNA sequences of eight of these mutants isolated after T3showed a single base change, a G-to-A transition, at position6256 (Fig. 2). We assume that this mutation originated fromthe ss O6MeGua-M13mp8 genome in which the adduct was

located in the (-) strand (Fig. 1). This mutation created anamber codon in the lacZ' gene that apparently was partiallysuppressed in JM103 cells (a supE strain), resulting in light-blue plaques. These mutants, originating from the adduct inthe (-) strand, are designated Mlb.The final mutant class observed was a four-base deletion,

which as indicated below apparently was not caused byO6MeGua. These mutants produced white plaques on

IPTG/X-Gal plates. The DNA from 10 of these plaques was

sequenced, and all had undergone deletion of the central fourbases of the Pst I recognition site. These phage producedwhite plaques, because this mutation shifted the readingframe of the lacZ' gene. These mutants probably arose dur-ing construction of O6MeGua-M13mp8, possibly from blunt-end-duplex (11) or gapped-duplex (15) ligation. As indicatedin the next section, these mutants were of value as an inter-nal standard used to calculate MF. They are designated Mw.MF of 06MeGua. The following conditions must be met in

order to calculate the, MF of O6MeGua by the plaque-colormethod, which was the principal method used. (i) The meth-od assumes that the observed base-pair substitutions were

targeted at the genome position that originally containedO6MeGua, specifically in the Pst I site. (it) The calculationsrely on the fact that virtually no wt phage survived the mu-

tant-selection scheme. This wa$ verified in that the mutant-selection protocol (steps 3 and 6) reduced the number of wtphage to less than 0.03% of the original level. (iii) It was

essential that the relative levels of the respective mutantclasses did not change through the mutant-enrichment pro-cedure. To test this, a mixture of phage consisting of 62%Mb, 13% Mib, and 25% Mw was taken through all of the pro-cedural steps between T2 and T3 (Fig. 1). After T3 the pro-portions of the three mutant species were again determinedby plating the phage mixtures and enumerating plaques ineach of the three color categories. It was found that the rela-tive amounts of the mutant species were virtually un-

changed, indicating a lack of bias; this was expected becausethese mutations occurred in an area of the genome that isirrelevant to phage viability (12). Additional support for thisresult came from evidence described below, indicating thatMFs calculated by the plaque-color method agree well withthose determined by two methods not requiring the mutant-enrichment protocol.The MF solely due to processing of the adduct in either the

(-) or (+) strand is, respectively, MF1 = M-/(Mjj + wtj) or

MF2 = Mb+/(Mb + wttD. We were unable to determine thesequantities, because our system did not allow us to distin-guish wt phage that arose from the (-) versus the (+) strand(wt- and wtb, respectively, where wtb - wtj + wt+). Thus,we have defined MF- (Eq. 1) and MF+ (Eq. 2), which repre-sent the fraction of the 06MeGua-Ml3mp8-derived phagepopulation produced after T1 that had mutations originatingin the (-) and (+) strands, respectively.

MF = lMb+(wtb + Mb + Mb)

MF+ =b(wtb + M+ + Mjl)

[I]

[2]

The following ratios (designated R1-R5) were used to cal-culate MF- and MF'. R1 and R2 were determined fromplaque colors produced after Ti, while R3, R4, and R5 weredetermined after T3.

light blue after T1= total blue after T1

R (light blue + white) after T12 total blue after T1

R - light blue after T3- (light blue + white) after T3

dark blpe after T3light blue after T3

Mlbwtb + M; + Mlb

Mu, + Mwwtb + Mb- + Mlb

M,-blb + Mw

MiiMbM,-b

dark blue after T3 _ Mb(light blue + white) after T3 Mjj + MW

Using R1 through R5, MF- could be calculated accordingto either Eq. 3 or 4, and MF+ could be calculated by either

Eq. 5 or 6.

MF = RIMF- = R2 x R3MF+ = RI x R4MF+ = R2 x R5

[3][4]

[5][61

The values determined from Eqs. 3 and 4 for MF- (andEqs. S and 6 for MF+) were averaged.The sum of MF+ and MF- is defined as total MF (MFV),

and, as can be readily shown, the mutation frequency forO6MeGua must be equal to or greater than -MFt. Values forMFt, MF-, and MF+ are presented in Table 1 for the experi-ment described above. The mutation frequency, MFt, for

06MeGua in the Pst I site in vivo was determined to be 0.4%.Enhancement of 06MeGua-Derived Mutants in Repair-

Compromised Cells. The MF determined above was =2 or-

ders of magnitude less than that seen for 06MeGua in vitro

(16, 17). We suspected this difference was due to the pres-ence in E. coli of one of the repair proteins associated withthe adaptive response (8), O0MeGua MeTase, which re-

stores guanine in DNA at the site of an 06MeGua by trans-

ferring the methyl group to itself (18). This protein is irre-

versibly inactivated by alkylation (9, 10, 19), and we were

able to utilize this property to diminish intracellular O6Me-Gua-repair capacity. Two min before the 06MeGua-M13mp8uptake step in the transformation (Fig. 1, step 1), host cellswere treated with MNNG, which introduced 06MeGua resi-dues into the host chromosome (9, 10). Repair of these le-

sions depleted levels of active O6MeGua MeTase and, thus,

Biochemistry: Loechler et al.

6274 Biochemistry: Loechler et al.

Table 1. Percentage MF due to O6MeGua in O6MeGua-M13mp8O6MeGua-M13mp8 MF, %

MNNG Sequencingchallenge Plaque-color method* gel method M13mp8

jig/ml MF- MF+ MFt MF-t MFt, %

Experiment 10 0.08 0.36§ 0.4 0.6 c0.0317 1.3 4.1 5.4 1.5 0.1133 3.6 4.7 8.3 3.4 <0.2050 4.1 13.7 17.8 4.1 <0.16

Experiment 2§0 (no Pst I) 0.4 0.21 0.6 0.850 (no Pst I) 7.4 14.1 21.5 7.150 (with Pst I) 3.8 14.8 18.6 3.2

*The plaque-color method for determining MF is described in Re-sults.tThe sequencing gel method for determining the MF in the (-)strand (MF-) is described in Fig. 3.tUpper limits of the MFs of wt M13mp8 [viral (+) strand] controlswere determined slightly differently than for 06MeGua-Ml3mp8.The same amount of RF DNA was used for all samples in step 6 ofFig. 1. After T3, the number of dark-blue plaques observed fromcontrol M13mp8-derived material was divided by the number ofdark-blue plaques from the O6MeGua-M13mp8-derived material.This value, when multiplied by the mutant fraction of 06MeGua-M13mp8, gave the upper limit of MF of control DNA.In experiment 2, ds 06MeGua-Ml3mp8 was either treated (withPst I) or not treated (no Pst I) with 25 units of Pst I prior to denatur-ation in 0.1 M NaOH and transformation (see Fig. 1).1This number was estimated as follows. In the absence of MNNGchallenge, the apparent MF in the (+) strand was 0.50%. However,the DNA sequencing results indicated that only 10 out of 14 (71%)of these mutants were O6MeGua-induced. Thus, MF+ in the ab-sence of MNNG challenge is (0.50% x 0.71 =) 0.36%. A similarcorrection was applied in experiment 2 to the data from unchal-lenged cells.

diminished the ability of cells to repair the adduct in O6Me-Gua-Ml3mp8. [MNNG treatment is known to induce severalDNA repair systems (8, 20); however, we assume that copy-ing of the O6MeGua lesion, estimated to take <2 min (21,22), would occur prior to their induction, making their effecton the mutagenicity of O6MeGua insignificant.] The resultsof this experiment for challenges with increasing concentra-tions of MNNG are presented in Table 1 (experiment 1). Asshown, the mutant fraction increased with the level ofMNNG treatment, presumably because a progressivelygreater fraction of the O6MeGua MeTase proteins in the cellwas inactivated. At the highest level ofMNNG treatment (50pg/ml), MFt had increased almost 50-fold over the compara-ble value in unchallenged cells. As a control, E. coli cellschallenged identically with MNNG were transformed withwt M13mp8 DNA [viral (+) strand]. Mutation of this DNA toPst I insensitivity was insignificant, which ensured that themutants derived from O6MeGua in the (+) strand did notarise from alkylation of the viral DNA during the MNNGchallenge. It is assumed that the same would be true for the(-) strand, but this was not tested.MF+ consistently was -3-fold higher than MF-. Two pos-

sible explanations for this observation are either that the (+)strand is more transfectable (14) or that the replication appa-ratus encounters the lesion in the (+) strand sooner than inthe (-) strand. The (+) strand is the normal replication pre-cursor (21); thus, infected cells possibly initiate DNA syn-thesis more efficiently on this strand.While the frequency of Mb and Mb increased as the intra-

cellular level of O6MeGua MeTase was depleted, the fre-quency of the four-base and -30-base deletions did not (datanot shown). This was taken as evidence that these deletions

were by-products of the genetic engineering scheme used toconstruct 06MeGua-Ml3mp8 and that their existence wasnot related to mutagenic processing of 06MeGua.To determine the MF due solely to 06MeGua, it was nec-

essary to investigate whether the O6MeGua-M13mp8 used inthe first transformation (Fig. 1, step 1) was contaminatedwith DNA containing the wt sequence at the Pst I site. Inprevious work, we found that having O6MeGua at this sitestrongly inhibited the cleavage of 06MeGua-Ml3mp8 by PstI (11). Thus, Pst I treatment would not cleave this DNA,although it would linearize any contaminating wt materialand, thereby, essentially eliminate its biological activity. Ex-periment 2 of Table 1 shows that the MF did not increaseafter Pst I treatment prior to T1. An unexpected finding wasthat one data point, MF-, decreased upon Pst I treatment; atpresent the mechanistic significance is unknown. When thedata are viewed as a whole, however, the general conclusiondrawn from this experiment and from other data (11) is thatO6MeGua-Ml3mp8 was essentially free of wt DNA.Confirmation of the Method Used to Determine the MF. By

two methods, it was possible to corroborate some of the MFvalues calculated by the plaque-color method describedabove. Both methods utilized phage from T1 (i.e., prior tothe mutant enrichment procedure). With the first method,phage (experiment 2, Pst I-treated sample) were plated, andthe DNA arising from 27 dark-blue plaques was sequenced.This plaque-color category was expected to include (+)-strand mutants (Mt) and wt phage (wtb). DNA sequenceanalysis of the individual phage showed that four plaqueswere mutant, while the remainder were wt. The MF+ deter-mined by this method was 15% (4/27 x 100), which is inagreement with the value reported in Table 1 (14.8%).For the second method, ss DNA was isolated from all of

the phage samples listed in Table 1, and the "adenine" DNAsequencing reaction was performed. [Each sample con-tained, in various proportions, (-)- and (+)-strand mutants,the four-base deletion mutant, and wt DNA]. In the autora-diogram of the DNA sequencing gel from these reactions(Fig. 3A), only the (-)-strand mutant could contribute to anadenine band at position 6256 because DNA from the otherthree species all have guanine at that site. Significantly, theintensity of this band reflected the fraction of phage in themixture that was (-)-strand mutants; thus, scanning densi-tometry provided a means to calculate MF-. The details ofthis procedure are presented in Fig. 3, and again the results(Table 1) agree well with those determined by the plaque-color method.

DISCUSSIONIn order to attribute the mutations of a DNA-damaging agentto a specific DNA lesion, it is essential to show that the le-sion can cause or contribute to the pattern of mutagenesisobserved in cells treated with that agent. The G-to-A transi-tion seen here for O6MeGua is the predominant base-pairingmutation seen in cells treated with alkylating agents (23), andit is the mutation predicted from model building (5) and nu-merous in vitro studies (16, 17, 24, 25). In addition, our re-sults showed that 06MeGua-specific mutagenesis increasedmarkedly in cells in which MNNG had reduced repair capa-bility for this lesion. Again this is in accord with what hasbeen observed with alkylating agents, in that treatments thatare known to reduce the level of O6MeGua MeTase result inelevated levels of both genomic 06MeGua and mutation (9).The latter observation has been used to support the argu-ment that 06MeGua is responsible for the mutagenic effectsof alkylating agents, although it cannot be ruled out that oth-er adducts show the same correlation. Thus, while the cur-rent observations alone do not prove that 06MeGua is re-sponsible for alkylating agent-induced mutagenesis, they do

Proc. NatL Acad Sci. USA 81 (1984)

Proc. Natl. Acad. Sci. USA 81 (1984) 6275

A EXP EXP 2 B STANDARDSg/ml pLg/mI %(-) StrandMNNG; MNNG + Mutant

0 17 3350 0 50 50 0 2 5 10 15 20

m~w.s_ _

62546256

_c

O-

9__

5 10 15

%(-) Strand Mutant

FIG. 3. Estimation of the MF in the (-) strand from DNA se-

quencing gels. Phage from T1 for each of the seven samples listed inTable 1 were amplified in JM103, and ss DNA was isolated. In paral-lel, standards were generated by mixing known amounts of purified,(-)-strand mutant DNA with wt DNA (final concentration of (-)-strand mutant; 0, 2, 5, 10, 15, and 20%). Samples were adjusted tothe same DNA concentration, and the "adenine" sequencing reac-

tion was performed. After polyacrylamide gel electrophoresis, thegels were autoradiographed. Representative autoradiograms of theseven samples in Table 1 and the standards are shown in A and B,respectively; the sample treated with Pst I prior to transformation(Table 1, experiment 2) is indicated by 50+. Only the (-)-strand mu-tant gave an adenine band at position 6256, whereas all other DNAspecies in the mixture except the four-base deletion mutant gave an

adenine band at position 6254. The autoradiograms were scannedwith a densitometer, and the relative intensity of the adenine band at

position 6256 to the adenine band at 6254 was computed. (The 6254band intensities were determined from films exposed for a muchshorter period of time because this band was much more intensethan the 6256 band.) This normalization essentially eliminated anysampling errors that could have accumulated during the procedure.[For the samples from experiments 1 and 2 (Table 1), the normaliza-tion is not completely accurate because the four-base deletion mu-

tant does not contribute to the adenine band at position 6254. Acorrection was applied to account for this small (<5%) effect.] (C)Standard curve; plot of the relative intensities of the standardsagainst the percentage of (-)-strand mutant. MF- for the samples inexperiments 1 and 2 were interpolated from this curve. Determina-tion of the MF+ was not possible by this method, since the four-basedeletion mutation as well as the (+ )-strand mutant gave a band in theT lane at position 6255, and wt DNA produced a shadow at thisposition (Fig. 2).

show that this lesion, in isolation from all other alkyl ad-ducts, is sufficient to cause a pattern of mutagenesis that isqualitatively and quantitatively identical to that caused byalkylating agents.

This study on the mutagenesis of a carcinogen built into a

known site in a genome has shown both the advantages andlimitations of such an approach. One of the problems en-

countered was the fact that side-product deletions unrelatedto the presence of the chemical adduct were created. Al-though these deletions occurred at a low frequency, theycould complicate the interpretation of mutagenesis data on

other adducts that exhibit very low mutagenic efficiency or

specifically induce deletions. Our approach also requiresthat the carcinogen is built into the viral genome as a small,modified oligonucleotide, and a potential problem is thatbulky adducts may inhibit the requisite ligation step. Shouldthis problem arise, one solution would be to synthesize larg-er oligonucleotides that can be ligated into genomes withlarger gaps. Finally, by analogy with other ss viruses (21,

22), it was thought that the adduct-containing (-) strandwould give rise to few phage if any. We found unexpectedlythat the (-) strand was a significant source of mutants andfound that having a mixture of mutants from the (+) and (-)strands made calculation of MF complicated.There are also several noteworthy characteristics of this

approach to genotoxicity measurements. These include thefact that the host cell need not suffer DNA damage, becausethe dose of mutagen is built into an extrachromosomal ele-ment as a single adduct. Hence, it is possible to make muta-genesis measurements when DNA repair systems are fullyactive, even those that are easily saturated. In addition,DNA vectors containing known adducts could be introducedinto host cells with specific defects in DNA repair; suchstudies would be directed at identifying the genes involved inthe processing of known DNA lesions. Finally, using DNAvectors based on eukaryotic viruses, the same approach asdescribed here could be used to investigate patterns of muta-genesis of DNA adducts in mammalian cells.

For their contributions to this work, we express our gratitude toG. H. Buchi, M. Chow, L. B. Couto, M. Ellison, K. W. Fowler,and D. D. Lasko. Financial support was provided by Grants 5 P01ES00597, T 32 ES07020, and CA 33821 and by the Monsanto Fund.

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