Nucleic Acids Research, 1993, Vol. 21, No. 16 3755-3760

Site-specific mutagenesis induced by single06-alkylguanines (06-n-propyl, 06-n-butyl, 06-n-octyl) in vivo

Petra M.Baumgart, Hans-Christian Kliem, Jutta Gottfried-Anacker', Manfred Wiessler andHeinz H.Schmeiser*Department of Molecular Toxicology, German Cancer Research Center, 69009 Heidelberg, Germany

Received April 22, 1993; Revised and Accepted June 28, 1993

ABSTRACTThe mutagenic activity of a series of longer chain06-n-alkylguanine residues (06-n-propyl, 06-n-butyl,06-n-octyl) has been analyzed using a plasmidmolecule (pUC 9) in which single 06-alkylguanineswere positioned in the unique Pstl recognition site byshot gun ligation (Nucleic Acids Res. 13, 3305 - 3316(1985)) of overlapping synthetic oligonucleotides. Aftertransfection of these vectors into E.coli cells havingnormal DNA repair systems, progeny plasmids wereproduced, of which 2.6%, 2.8% and 4.3% weremutated in their Pstl site when containing 06-n-propylguanine, 06-n-butylguanine, 06-n-octylguanine,respectively. DNA sequence analysis of mutant plasmidgenomes revealed that 06-n-propylguanine and 06-n-butylguanine Induced exclusively G - A transitionslocated specifically at the preselected site. 06-n-octylguanine induced apart from G - A transitions(70%) also targeted G -T transversions (30%). Theseresults indicate that the mutation frequency of longerchain 06-alkylguanines can be substantial in cells withnormal repair systems and that the mutation patterndepends on the nature of the alkyl group.

INTRODUCTIONThe formation of covalent DNA adducts is thought to be oneof the critical steps in the process of chemical carcinogenesis (1).As carcinogens produce multiple DNA adducts within thegenome, it is difficult to determine the contribution of specificadducts to biological end points, such as mutagenicity (2).

It is the purpose of site-directed mutagenesis studies (3) toexplain how individual chemical lesions formed in DNA bycarcinogens are converted into mutations found in target genesof carcinogenesis, e.g. cellular oncogenes (4). These studies onpremutagenic lesions have advanced recently due to thedevelopment of techniques to construct DNA vectors that containadducts of defined chemical structure at defined sites of the vector(5). The general strategy of these studies was to synthesize asingle-stranded oligonucleotide that contains a specific adductwhich is subsequently incorporated into an appropriate vector.

This is accomplished by the construction of gapped duplexespioneered by Essigmann et al. (6).

06-alkylguanine and 04-alkylthymine are believed to play akey role in the initiation of cancer by alkylating agents (7,8).The mutagenic properties of these miscoding alkylbases have beendemonstrated by experiments on replication of synthetic DNAtemplates (9), DNA sequencing after exposure of cells toalkylating compounds (10,11) and more recently by site-directedmutagenesis studies (6,12). These studies were performed mainlywith methylated and ethylated bases the mutagenic actvity ofhigher alkylated bases have been examined by a lesser extent.In particular, the 06-alkylguanines have been suggested torepresent premutagenic lesions for the mutagenic and car-cinogenic effects exerted by alkylating agents (13,14). Indeed, theformation of 06-methylguanine by the carcinogen, N-methyl-N-nitrosourea, has been correlated with the activating mutationsfound in the H-ras oncogene of rat mammary tumors inducedby this alkylating agent (15, 16).

Likewise, the carcinogenicity and mutagenicity of N-nitrosocompounds, representing an important group of alkylating agentsis also believed to result primarily from alkylation of 06 ofguanine (17). Expressing the relative extents of reaction atdifferent sites of guanine as the ratio of 06/N-7 alkylation,nitroso compounds with longer alkyl chains produce relativelymore 06-alkylation product with an 06/N-7 ratio of 0.6-0.7compared to an 06/N-7 ratio of 0.11 for methylating N-nitrosocompounds (9). The dialkylnitrosamines, di-n-propylnitrosamineand di-n-butylnitrosamine are potent carcinogens in rats, bothinducing liver tumors on oral administration. Following s.c.injection of di-n-butylnitrosamine the urinary bladder is the maintarget organ (18). Di-n-butylnitrosamine has also been identifiedas an environmental contaminant in the working places of therubber and tyre industry (19) and in baby bottle nipples (20).These nitrosamines are thought to produce 06-n-propylguanine(06-n-propyl Gua) or 06-n-butylguanine (06-n-butyl Gua) apartfrom other modifications in DNA like rearrangements of the alkylgroup as shown by the corresponding N-alkyl-N-nitrosoureas(21, 22). These considerations prompted us to study 06-n-propylGua and 06-n-butyl Gua in terms of mutagenic potency.Additionally, the bulkier 06-n-octylguanine (06-n-octyl Gua)

* To whom correspondence should be addressed+ Present address: Department of Chemistry, Food Chem. & Env. Tox., University of Kaiserslautem, 6750 Kaiserslautern, Germany

.j- 1993 Oxford University Press

3756 Nucleic Acids Research, 1993, Vol. 21, No. 16

was included to assess the impact of the increasing chain lengthof the 06-substituent on the mutagenic potency of the modifiedguanine.To investigate the mutagenic activity of a series of longer

chain 06-alkylguanines in E. coli cells with normal repair,we constructed a double stranded vector in which the06-alkylguanines were situated at a preselected site by shot gunligation of overlapping synthetic oligonucleotides. Followingtransfection and replication of these vectors in E. coli, mutantprogeny was analyzed to determine the relative mutationfrequencies and mutational specificities of the three DNA adductsin the same sequence context and the same test system. It wasalso our objective to compare our results obtained with 06-n-propyl Gua and 06-n-butyl Gua with those reported byChambers and coworkers (12, 23) obtained in a different testsystem.

MATERIALS AND METHODSReagents. 5-Bromo-4-chloro-3-indolyl-f3-D-galactopyranoside (X-gal) was supplied by Serva and isopropyl-,B-D-thio-galactopyranoside (IPTG) from BRL. [-y-32P]ATP (7000Ci/mmol) was bought from ICN Biomedicals GmbH and[a-35S]ATP was obtained from Amersham.

Enzymes. The restriction enzymes, spleen phosphodiesterase, T4polynucleotide kinase were obtained from Boehringer Mannheim.The micrococcal endonuclease, bacterial alkaline phosphatasewere from Sigma, venom phosphodiesterase from Merck andT4-DNA ligase from Biolabs. These enzymes were used asrecommended by the manufacturers or as described below.

Vector and bacteria. The plasmid pUC9 was bought from Sigmaand E.coli strain BMH 71-18 [supE thi A (lac-pro AB) F'(proAB+ laqIq lac Z AM15)] was taken from the site directedmutagenesis kit from Boehringer Mannheim. E. coli strain BMH71-18 is derived from E. coli K12 and is believed to have normalDNA repair systems.

Synthesis ofoligonucleotides. The 06-allylated deoxyguanosineamidites were prepared as described (24). 5'-O-Dimethoxytrityl-N2-phenoxyacetyl-deoxyguanosine was alkylated by reactionwith an etheral solution of the appropriate diazoalkane intetrahydrofuran. Products were phosphitylated with2-cyanoetlyldiisopropylamido-chlorophosphit according todescribed methods (25).The modified lOmers 5'-d (AGATCT*GCAG)-3' (with the

06-n-propyl-, 06-n-butyl-, 06-n-octyl Gua adducts in the PstIrecognition sequence, G* representing either 06-n-propyl Gua,06-n-butyl Gua or 06-n-octyl Gua), the unmodified lOmer andthe complementary 20mer 5'-d (AATTCTGCAGATCTCGAG-AA)-3' were synthesized on a 0.2jsmol scale using solid phasephosphoramidite chemistry on an Applied Biosystem, Inc., Model380A, DNA-synthesizer.

Characterization of oligonucleotides. Unmodified oligo-nucleotides were deprotected and purified by NENSORB puri-fication cartridges (DuPont NEN) as described by the supplier.Modified oligonucleotides (lOmers) were cleaved from thecolumn solid support by the standard concentrated NH40Htreatment and purified by HPLC as described (26). Afterdetritylation with 80% acetic acid the modified and unmodified

lOmers were identified and purified by HPLC analysis as shownin Fig. 1.

Analysis ofbase composition. The 06-alkylated deoxyguanosinesused as standards for base composition analysis were preparedas described (27). 06-isopropylguanine and 06-isobutylguanine,possible side reaction products from alkylation, were alsoincluded in the analysis. Base composition analysis was performedas described (10). The amount of each nucleoside was determinedby comparing the integrated absorbance at 260 nm with theabsorbance of standard solutions. The chromatograms confirmedthe base ratio expected and indicated that these oligonucleotidesamples are free of detectable contamination by the diaminopurinenucleoside, the product of ammonolysis of 06-substituted2'-deoxyguanosine derivatives (24), which eluted at 8.3 minbetween 2'-deoxythymidine and 2'-deoxyadenosine under theconditions used.

32P-postlabeling analysis. The purity of modified and un-modified oligonucleotides was analyzed by the standard versionof the 32P-postlabeling assay according to Gupta et al. (28). 5',3 '-deoxynucleoside bisphosphates were resolved bychromatography in directions Dl and D2 by 0.28 M ammoniumsulfate, 50 mM NaH2PO4 pH 6.5, as shown in Figure 2. Afterautoradiography spots were excised and radioactivity wasdetermined by Cerenkov counting as described (29).As shown in figure 2 the modified 06-n-butyldeoxyguanosine

bisphosphate migrated between the deoxyguanosine bisphosphateand the deoxyadenosine bisphosphate. 06-n-propyldeoxy-guanosine bisphosphate migrated close to deoxyadenosine bis-phosphate whereas 06-n-octyldeoxyguanosine bisphosphatestayed at the origin (data not shown).

5'-Phosphorylation of oligonucleotides. The two lOmer oligo-nucleotides 5'-d (AGCTTTCTCG)-3', 5'-d(AGATCT*GC-AG)-3' and the complementary 20mer 5 '-d(AATT-CTGCAGATCTCGAGAA)-3' were 5'-phosphorylated in onestep. 0.5 mg of each lOmer and 1gg of the 20mer were incubatedfor 1 h at 37°C with 5U T4 polynucleotide kinase and['y-32P]ATP. The enzyme was inactivated at 95°C, the DNAduplex was precipitated with ethanol, dried and resuspended asdescribed (30).

Construction ofsite-specifically modifiedplasmids pUC9-1. Theprotocol for preparation of the site-specifically modified plasmidsis outlined in Figure 3. Plasmid pUC9 (5jig) was digested withHindIII (25U) and EcoRI (32U) to remove a 30bp fragment ofthe endogenous polylinker. The incubation mixture was extractedwith phenol/chloroform and DNA was precipitated according toManiatis et al. (26) probably removing most of the 30bp DNAfragment. The pellet was dried and resuspended in TE-buffer.The resulting linearized plasmid carried noncomplementaryprotruding termini which were used to ensure the orientation ofthe insertion as well as to prevent recircularization of the plasmid.Therefore dephosphorylation of the cleaved plasmid was omitted(31). The ligation reaction was performed according toGrundstrom et al. (30) with minor modifications, 0.1 pmollinearized plasmid (1.45/1) and 1.5pmol of phosphorylatedoligonucleotide mixture (lOIul) were incubated with 200 UT4-DNA ligase at 13°C for 30 min. Ligase was inactivated byincubation at 70°C (10 min). The mixture was digested withBamHI (5U) to cut such plasmid molecules which had inserted

Nucleic Acids Research, 1993, Vol. 21, No. 16 3757

the previously excised 30 bp polylinker fragment. Aliquots ofthese digests were directly used for transformation of E.coli.

Transformation of E.coli cells. Digests of pUC9-10, preparedas above, were transfected into competent E.coli BMH 71-18cells according to standard protocols (31). Bacterial mixtures weregrown in LB medium in the presence of ampicillin (50%g/ml)at 37°C overnight (transformation efficiency 108/pug plasmid).Bacterial colonies that contained recombinant plasmids were

identified by a-complementation (31, 32). The presence of theinsert was confirmed by the appearance of white colonies on

indicator plates as opposed to the dark blue phenotype of the wild-type pUC9 DNA. White colonies were picked and grown

overnight at 37°C in LB medium with ampicillin.

Enrichment of mutant plasmids resistant to PstI cleavage. Thestrategy for enrichment of mutant plasmids is outlined inFigure 4. After the first transformation of E. coli BMH 71-18plasmid DNA was isolated as previously described (31). Isolatedplasmids were digested with PstI according to the protocol ofthe supplier; this treatment linearized wt pUC9-10 molecules butleft mutants intact. The mixture was loaded on a 1 % agarosegel, linearized molecules were separated from supercoiledplasmid molecules by gel electrophoresis and the bandrepresenting intact plasmids was cut out.The plasmid DNA was eluted from the gel slice by the Biotrap

electro-separation-system (Schleicher and Schuell) as describedby the manufacturer. This isolated plasmid DNA was used foranother round of transformation of E. coli BMH 71-18 asdescribed above and plated on indicator plates. White colonieswere picked and plasmid DNA was isolated from individualcolonies as described above.

Identification of mutations. Plasmid DNA isolated from singlewhite colonies was analyzed by digestion with PstI and XhoI toensure that plasmid preparations contain mutations in the PstIsite as well as the insert was incorporated correctly. Onlypreparations resistant to PstI and susceptible to Xhol digestionwere sequenced by the chain-termination method described bySanger et al. (33). Sequencing reactions were performed withthe T7 sequencing kit from Pharmacia on ds plasmid DNA.

Mutation frequency (MF) determination. To determine themutation frequencies of 06-n-propyl Gua, 06-n-butyl Gua and06-n-octyl Gua aliquots of plasmid DNA's isolated aftertransformation of E. coli BMH 71-18 were digested in paralleleither with PstI, XhoI or SspI and used to transform E. coli BMH71-18 for a second time. Briefly, 20ng of the respective plasmidswere incubated in parallel with PstI (3U) or XhoI (75U) as

described by the manufacturer for 1.5 h. To ensure completedigestion new enzyme was added to each mixture and incubatedfor another 1.5 h. The resulting mixtures were used to transformcompetent E. coli BMH 71-18 and seeded on plates containingampicillin, IPTG and X-Gal, as described above. The resultingwhite colonies were counted and used for the calculations. Thetotal number of colonies was determined in parallel incubationsbut without enzyme addition and MFs were calculated from theaverage of three determinations as described in Results. In controlincubations with SspI we showed that linearized plasmid DNAis incapable of transforming E. coli BMH 71-18 (no transformantswere ever obtained).

'II'D

I1..

10 20 30Minutes

40 50

Figure 1. HPLC chromatograms of unmodified and 06-n-alkyl-modified decamer5'-d(AGATCT*GCAG)-3'. Analysis was performed on a Beckman UltrasphereODS column 4.6mmx25cm (5,gm particle size) eluted with a linear gradient of7-17% acetonitrile in 0. IM triethylammonium acetate (pH 7) over 50 min ata flow rate of lml/min.

RESULTSOligonucleotide synthesis and characterization. The 06-n-propyl-, 06-n-butyl- and 06-n-octyldeoxyguanosine amiditeswere synthesized according to published procedures (24) and usedto synthezise the modified oligonucleotides by solid phasephosphoramidite chemistry. The fully deprotected 10mers were

analyzed by reversed phase HPLC (Figure 1) showing that anincrease in length of the 06-alkyl group results in an increasein retention time under the used conditions. Base compositionanalysis (34), (data not shown) and the more sensitive 32p-postlabeling assay (28), (Figure 2) were used to assess the levelof contamination of the modified oligomers. Both methodsshowed that these DNA samples were free of detectablecontaminations and therefore suitable for biological experiments.

Construction of pUC9 molecules containing single 06-alkyl-guanines situated at a defined site. The strategy used for theconstruction of vectors was twofold. Firstly to be suitable forrelatively short, modified oligonucleotides for insertion, sincesynthesis and purification of longer sequences by HPLC is moredifficult. Secondly, the modified base should be located in a PstIrecognition site to allow selection of resulting mutants by a simpledigestion step to miniimize the number of sequencing analysesof single clones.To this end we used plasmid pUC9 a commonly used plasmid

vector that permits histochemical identification of recombinantclones, developed by Messing et al. (35). A 30bp fragment,carrying an unique BamHI site, was removed from the polylinkerregion of pUC9 by digestion with HindIII and EcoRI. This

A

16.05 unmodificd l0mcr

B21.19 06-n-prop. nodifi-Ld lOncr

C

24.27 o6-n-but. modifid l0mcr

6- n - >t. iiioJiilUl lOnwr 45.93

3758 Nucleic Acids Research, 1993, Vol. 21, No. 16

tii

(L IIEo' amNin r

Hindlll EacS&mH I

*ljii;-

CA

,

))+ Hind!II ll+ EcoRI (

Hindlll EcoRI +T4DNA

I: . . . :*RI .

Phosphorylatedoligonucleotides

TTCTC AGATCTCAGEG IHidl! |AGAGCTCTAGACGTCT

L- Xhol Pst* u

Figure 3. Scheme for insertion of the two decamers d-(AGCTTTCTCG), d-(A-GATCT*GCAG) and the complementary 20mer d-(AATTCTGCAGATCTCG-AGAA) by shot gun ligation of overlapping synthetic oligonucleotides into thepolylinker of pUC 9. *G represents either 06-n-propyl Gua, 06-n-butyl Gua,06-n-octyl Gua or guanine. Correct insert alignment creates an unique PstI siteand an unique X1ol site. Ligation resulted in the insertion mutant pUC9-10introducing a +2 frameshift in the Lac Z gene of pUC9.

PU-10 i-~~~~~~~~~PstiPlasmid DNA - white+BamHI 2Transformation Colonies+ Transformation

of E coli (BMH 71-18)

Hind!II EcoR I

Figure 2. Autoradiographs of 32P-labeled digests of (a) decamer 5'-d(AGAT-CTGCAG)-3' and (b) 06-n-butyl Gua modified decamer 5'-d(AGATCT*GC-AG)-3'. pdGnBp, 06-n-butyldeoxyguanosine bisphosphate. For assay descriptionsee Material and Methods.

resulted in the formation of noncomplementary protrudingtermini, thereby preventing efficient recircularization of thelinearized vector as well as the insert containing compatible endsis built in preferably in one direction (35).

Insertion of the oligomers carrying site specifically modifiedguanines was accomplished by a method (shot gun ligation)described by Grundstrom et al. (30). The two 10meroligonucleotides 5'-d(AGCTTTCTCG)-3', 5'-d(AGATCT*GC-AG)-3' (*G represents 06-n-propyl-, 06-n-butyl- and 06-n-octylGua) and the complementary 20-mer 5'-d (AATTCTGCAGAT-CTCGAGAA)-3' (Figure) were combined, 5'-phosphorylatedand after the addition of the linearized vector ligated by T4 DNALigase at 13°C for 30 min. Analysis of the ligation reactionson 1% agarose gels confirmed results described by Koehl et al.(36), that longer incubation times had little effect on the reactionproduct. The successful integration of a 20bp insert formed bythe correct assembly of the three overlapping syntheticoligonucleotides introduced a +2 frameshift in the Lac Z geneof the vector resulting in the formation of an inactivated oa-peptideof ,B-galactosidase (31, 32). These insertion mutants form whitecolonies in E.coli BMH 71-18 cells in the presence ofIPTG/X-gal.

Characterization ofmutants. The protocol for selection of mutantplasmids is outlined in Figure 4. Aliquots of the ligation reactionwere digested with BamHI to linearize recircularized parent pUC9preventing transformation. The mixture was directly used totransform competent E. coli BMH 71-18 cells. After this first

Figure 4. Outline of procedure used for identification of 06-alkylguanine inducedmutations. Aliquots of ligation reactions, which produced pUC9-10 molecules,were digested with BamHI to linearize parent pUC9. Digests were directly usedto transform E.coli BMH 71-18. After isolation of plasmid DNA mutant plasmidswere enriched by PstI digestion. PstI digests were analyzed on agarose gelelectrophoresis. Intact supercoiled plasmid molecules were excised and used totransform E.coli BMH 71-18. White colonies were formed on indicator plates.Single white colonies were picked, amplified and sequenced.

round of transformation primarily white colonies appeared uponplating of cells on indicator plates. Some of the few light blueand blue colonies were analyzed and shown to contain plasmidsthat did not harbour the expected 20bp insert or the previouslyremoved 30bp insert. On the contrary, only fragments of the 20bpinserts could be detected (data not shown) but showed no evidencefor possible frameshift mutations in the polylinker region.For selection of mutated plasmids within the PstI site, originally

carrying the modified guanine bases, plasmid DNA preparationswere digested with PstI. The method used was introduced byLoechler et al. (37). To differentiate mutant and wild typepUC9-10 based on the fact that the progenitor plasmid DNAmolecules contained the adduct in the unique PstI site. Mutationsoccuring in this site render the plasmid DNA insensitive tocleavage by this endonuclease. This property made it possibleto isolate a pure mutant plasmid population for DNA sequencingand for calculation of MFs. After gel electrophoretic separationof plasmid preparations bands of supercoiled and linearizedmolecules were located by parallel separations of aliquots stainedby ethidiumbromide and the supercoiled bands were excised andeluted. In plasmid preparations obtained by transformation withpUC9-10 carrying the unmodified insert no supercoiled plasmidband was detectable, indicating that virtually no mutations wereinduced in the PstI site in the controls. These non PstI digestableplasmids were used to transform E. coli BMH 71-18 cells for asecond time and plated on IPTG/X-gal containing plates.

HindI11 EcoRI

*1 .f

.nBp

Nucleic Acids Research, 1993, Vol. 21, No. 16 3759

-- I IIIII~~~~~~~~~~11 IV

Figure S. Agarose dye gel for analysis of plasmid preparations. Plasmidpreparations (1) of parent plasmid pUC9 (2), (3), (4) of single white coloniesobtained after PstI digest enrichment were (a) undigested, (b) digested with X7wlor (c) digested with Pstl. L, molecular weight marker, X Hindm digest; I, relaxedcircular form; II, nicked form; HI, linear form; IV, supercoiled form.

G A T C

_T_"-~~~~_u~ A

T_m T

AA

G

A

_ CG

_T_ C

. TA_ ~ ~ ~~G

e _ ~~~~T

.. TT

_G_ ~~~~~A

A

G A T CGGT

. C'*g A

TT

"%& AAG

-"NW A

A4**M T

C

TAGAGC

TC

TT

C

GA

.qxw AC

C

B

G A T C

eV on;-_-

TAAGmm A

_ C

TT

_ A

_m G

4 Cam T

4 C_W T_o T

-U,

c

Figure 6. DNA sequences of insert regions of wild type and 06-alkyl Guainduced plasmid mutants. DNA of plasmids was isolated and sequenced asdescribed in Material and Methods using a primer complementary to the (-)plasmid strands. (A) Plasmid DNA of transformants, which were obtained afterligation of unmodified oligonucleotides and selection by a-complementation, (B)plasmid DNA of 06-n-propyl Gua or 06-n-butyl Gua induced mutants whichwere obtained after PstI digestion and selection by a-complementation, (C) plasmidDNA of 06-n-octyl Gua induced mutants which were obtained after PstI digestionand selection by a-complementation. 30% of plasmids analyzed contained theGC - TA transversions shown. Mutations are indicated by arrows.

Only white colonies were formed which were randomly pickedand amplified overnight. After isolation plasmid DNAs wereanalyzed on 1% agarose gels by PstI digestion for mutants inthe PstI site and by XhoI digestion for carrying the correct 20bpinsert (Figure 5).25 PstI resistant and XhoI susceptible plasmid DNAs of each

06-alkyl Gua modified plasmid were sequenced by the dideoxymethod (33).As shown in Figure 6 06-n-propyl Gua and 06-n-butyl Gua

induced exclusively GC - AT transition mutations specificallyat the site where the adducts were located. Likewise, 06-n-octylguanine induced only targeted mutations but apart fromGC - AT transitions also some GC - TA transversions (7 outof 25) were detected.

Mutation frequencies (MFs) of 06-n-propyl-, 06-n-butyl and06-n-octyl Gua. After introduction of the site-specificallymodified vectors pUC9-10 into E.coli BMH 71-18 cells, amixture of wild type and mutant plasmids was produced by theendogenous replication and repair systems of the bacterial cells.The MF's of the 06-alkylguanines were expressed as thefraction of white colonies produced after the secondtransformation (Figure 4) by PstI digestion in relation to thenumber of white colonies produced when PstI digestion wasomitted. Since plasmids resistant to XhoI cleavage do not containthe correct insert the number of white colonies obtained afterX7hoI digestion was subtracted. For example, the number ofmutants scored for the 06-n-propyl Gua residue were as follows:90 white colonies after PstI digestion, 535 white colonies withoutPst digestion, 76 white colonies after XhoI digestion. (Numbersare the average of three determinations)The MF's induced by 06-n-propyl-, 06-n-butyl- and 06-n-

octyl Gua were determined to be 2.6%, 2.8% and 4.3%,respectively. The increasing order of MF's propyl < butyl <octyl corresponds with increasing chain length of the06-alkylguanines tested. The results are not in agreement withthe results reported by Chambers et al. (23) showing that ethyland methyl 06-guanine induced a much higher MF than n-propyl- and n-butyl-06-guanine in E. coli AB1 157.

DISCUSSIONThe mutagenic activity of a series of longer chain 06-n-alkylguanines was analyzed in a PstI sequence context usingmethods that would have allowed us to detect any base pairingmutation and certain frameshift mutations in the vicinity of theadduct location. This was accomplished by using the shot gunstrategy of overlapping synthetic oligonucleotides to situate theadducts at a defined site in the vector.

In our test system 06-n-propyl Gua and 06-n-butyl Guainduced GC - AT transition mutations exclusively, which arethe predominant mutations of alkylating agents in bacteria andmammalian cells (10, 38). These results are consistent with theresults obtained by Chambers et al. (23). Mispairing of 06-n-alkylguanine with thymine is thought to be the mutationalmechanism (13, 39, 40). 06-n-octyl Gua, which was includedin the study to analyze the impact of a much longer n-allyl chain,induced apart from GC - AT transitions approximately 30%GC - TA transversion mutations. The latter could result from06-n-octyl Gua being recognized as a noncoding lesion by DNApolymerase thus inserting adenosine opposite to the adduct. Thisis consistent with the results reported for other noncoding lesionsand supports the hypothesis that, in the absence of instructionfrom the template, DNA polymerases tend to insert adenosineopposite the noninstructional lesion (41). That this mechanismincreases when the adduct investigated is a more bulky one hasalso been shown by reports from Mitra et al. (42), who foundGC - AT transitions and GC - TA transversions producedby 06-benzyl Gua. Likewise, -2 frameshift mutations (36) canbe excluded, since such mutations restore the reading frame ofthe Lac Z gene and cause the colonies to be blue. No such bluecolonies were detected.The fact that all mutant plasmids obtained by the 06-n-alkyl

Gua residues contained only targeted point mutations is strongevidence that 06-n-alkyl Gua's represent the premutageniclesions responsible for the mutations found. It was hypothesizedby Basu et al. (40) that the stability of the 06-alkyl Gua:thymine

4

go

3760 Nucleic Acids Research, 1993, Vol. 21, No. 16

base pairs should be more dependent on the steric bulk of thealkyl group than in a 06-alkyl-Gua: cytosine pair. Consequentlythis should result in reduction in mutagenicity (G - A) as thesize of the alkyl group increases. Our results do not support thissuggestion because 06-n-propyl Gua, 06-n-butyl Gua and 06-n-octyl Gua induced approximately the same MF's ( - 3%) ofG -A transitions under the conditions used. In fact, it waspresumed by Swann (39), that in DNA replication the alignmentof the bases is more important than the hydrogen-bonded strengthof the base pairs. Thus, the mechanism for miscoding is explainedby the fact that the 06-alkyl Gua: thymine pairs have thealignment of Watson-Crick base pairs, while the pairs formedbetween 06-alkyl Gua and cytosine have a wobble alignment.As shown by other groups the mutation frequency for a variety

of bulky adducts has been estimated to be 1- 3% (43, 44). Hence,MF's of 2.6-4.3% for the propyl, butyl and octyl modifiedguanines lies in the same range showing that even a n-propylmodification at 06 of guanine has a mutagenic potentialcomparable to adducts formed by polycyclic aromatichydrocarbons or aromatic amines.The MFs of 06-n-propyl- and 06-n-butyl Gua of 2.6% and

2.8% obtained by us in E.coli BMH 71-18 cells with normalrepair are rather different to those results reported by Chambers(23) who found 1 % mutant frequency induced by 06-n-propyl- and 06-n-butyl Gua when built into the (X174 genome.The differences can be explained by several factors as discussedby Chambers (23): (i) effect of neighbouring bases on themutagenicity of the adduct, (ii) differing experimental systems.The most important difference presumably lies in the verydifferent experimental assays. It is clear, however, that longerchain 06-n-alkylguanines can produce specific targetedmutations at substantial frequency levels in cells despite theirability to remove these adducts from DNA.

Future studies with cells having a different repair status willgive some insights in the efficiency of repair of longer chain06-n-alkyl-guanines which are likely substrates for excision-repair pathways (45, 40). Such excision repair may becomeincreasingly important as the size of the alkyl group increases.

ACKNOWLEDGEMENTSWe wish to express our thank to Dr J.W.G.Janssen for adviceand help in sequencing analysis, Dr W.Reinig for providing someoligodeoxynucleotides, and Dr B.Kaina for reviewing themanuscript before submission.

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Proc. Natl. Acad. Sci. USA, 81, 13-17.7. Goth, R. and Rajewsky, M. F. (1974) Proc. Natl. Acad. Sci. USA, 71,

639-643.8. Singer, B. (1986) Cancer Res., 46, 4879-4885.9. Saffhill, R., Margison, G.P. and O'Conner, P.J. (1985) Biochem. Biophys.

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