The Role of Mutagenic Metal Ions in MediatingIn Vitro Mispairing by AlkylpyrimidinesOpinder S. Bhanot and Jerome J. SolomonDepartment of Environmental Medicine, New York University Medical Center, New York, New York

A variety of alkylating mutagens and carcinogens produce pyrimidine adducts in DNA that block DNA synthesis in vitro. Since DNA synthesis past thelesion is a necessary step to produce mutations, we investigated the role of the mutagenic metal ion Mn++ in facilitating DNA synthesis pastalkylpyrimidines. In the presence of the natural metal activator Mg++, N3-ethyldeoxythymidine (N3-Et-dT) and 02-ethyideoxythymidine (02-Et-dT), pre-sent at a single site in DNA, blocked in vitro DNA synthesis 3' to the lesion and after incorporating dA opposite each lesion. The presence of Mn++permitted postlesion synthesis with dT misincorporated opposite N3-Et-dT and 02-Et-dT, implicating these lesions in A.T->T-A transversion mutagen-esis. The DNA synthesis block by 04-ethyldeoxythymidine (04-Et-dT) in the presence of Mg++ was partial and was also removed by Mn++. Consistentwith in vivo studies, dG was incorporated opposite 04-Et-dT during postlesion synthesis, leading to A-T-*G-C transition mutagenesis. We also havediscovered a new class of DNA adducts, N3-hydroxyalkyldeoxyuridine (3-HA-dU) lesions, which are produced by mutagenic and carcinogenic aliphaticepoxides. 3-HA-dU is formed after initial alkylation at the N3 position of dC followed by a rapid hydrolytic deamination. As observed with the analo-gous mutagenic N3-Et-dT, the ethylene oxide-induced 3-hydroxyethyldeoxyuridine (3-HE-dU) blocked in vitro DNA synthesis, which could be by-passed in the presence of Mn++. The nucleotide incorporated opposite 3-HE-dU during postlesion synthesis is being identified. These studies suggesta role for Mn++ in mediating mutagenic and carcinogenic effects of environmentally important ethylating agents and aliphatic epoxides. - EnvironHealth Perspect 102(Suppl 3):81-90 (1994).

Key words: alkylating agents, alkylpyrimidines,pounds, DNA polymerase

DNA adducts, divalent metal activator, misincorporation, DNA synthesis, mutation, nitroso com-

Introduction

Alkylating agents have been used exten-

sively in studying the mechanisms of muta-genicity and carcinogenicity (1-8), becauseof their ability to react with DNA either invitro or in vivo. Of the best characterizedalkylating agents are the N-nitroso com-

pounds. Their occurrence is widespread inthe environment, and human exposure

from natural and pollutant sources is uni-versal (9). These agents induce tumors in a

wide variety of tissues of different animalspecies (5,10-14) and probably humans(9,15).

Most N-nitroso-alkylating agents medi-ate their biological activities in part by

This paper was presented at the Second InternationalMeeting on Molecular Mechanisms of Metal Toxicityand Carcinogenicity held 10-17 January 1993 inMadonna di Campiglio, Italy.We thank Dr. Peter C. Grevatt and Ms. Jean M.

Donahue for their participation in the DNA replicationstudies of ethyldeoxythymidine adducts. Theresearch described here was supported by researchgrant DMB-8607556 (OSB) from the NationalScience Foundation, grant ES 05694 (JJS) from theNational Institute of Environmental Health Sciences,grant CIAR 90-12 (JJS) from the Center for Indoor AirResearch, center grants CA 16087 and ES 00260from NIH, and grant SIG 9A from the AmericanCancer Society.

Address correspondence to Dr. Opinder S.Bhanot, Department of Environmental Medicine,New York University Medical Center, 550 FirstAvenue, New York, NY 10016-6451. Telephone (914)351-2204. Fax (914) 351-3489.

interacting with genomic DNA, formingcovalent adducts (4). Alkylation atthymine occurs at nucleophilic oxygensites, such as the 04- and &2positions ofthe base and at the N3 position (4,16).There are many factors determining thesensitivity to the toxic, mutagenic, and car-cinogenic potential of N-nitroso alkylatingagents. One of these factors is the capacityto repair alkylated DNA. A substantialbody of experimental evidence has indi-cated that it is not the initial level of alkyla-tion but the persistence (lack of repair) ofpremutagenic alkyl adducts in tissueswhich is of major importance in mutagene-sis and malignancy in specific organs. Thishas been demonstrated for 06-alkyl-deoxyguanosine (06-alkyl-dG) and 04-alkyldeoxythymidine (04-alkyl-dT), wherethe persistence of these lesions did correlatewith organotropic malignancy (17-20). Awide range of independent studies has indi-cated that the repair of ethyldeoxythymi-dine (Et-dT) adducts in mammalian cells isvery slow (21). These adducts are amongthe highly persistent DNA alkylation prod-ucts in both cultured mammalian cells andanimal tissues (19,22). The persistence ofpremutagenic Et-dT adducts wouldincrease the probability of mutation at A-Tbase pairs relative to G-C. This is consis-tent with the prevalence of transversionand transition mutations at A-T base pairs

following in vivo exposure of mice to N-ethyl-N-nitrosourea (ENU) (23).

The mutational spectra of ENU hasbeen reported in a variety of systems. In E.coli, ENU induced mainly G-C-oAT andA-T-*G-C transition mutations (24).Presumably, these mutations resulted fromthe unrepaired 06-Et-dG (2,25,26) and04-Et-dT (2,5,27) lesions, respectively, asa consequence of their capacity to mispairwith dT and dG, respectively, during DNAreplication. Under SOS-induction, ENUgenerated a large fraction (46%) of trans-version mutations at A-T base pairs in E.coli (28). In human cells, ENU produced asignificant number (29% or more) of trans-version mutations at the A-T base pairs(8,29) in addition to the same G-C->A-Tand A-T-*GC transitions observed in E.coli. In vivo exposure of mice to ENU pre-dominantly (94%) induced transversionand transition mutations at A-T base pairs(23). A study of mutational spectra inSalmonella attributed transversions at A-Tbase pairs to Et-dT adducts (6).Transitions at A-T base pairs can resultfrom 04-Et-dT. The Et-dT lesions respon-sible for transversion mutations at A-Tbase pairs are not known.

The biological importance of A-Ttransversion mutations has been demon-strated in mammalian systems. TheA*T->T-A transversion event has beenproposed to account for two mutations of

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the mouse a- and [-globin genes arising inthe progeny of ENU-treated female mice(31,32). Tumors of the nervous systeminduced by transplacental treatment of ratswith ENU contained the neu oncogeneactivated by an AT-4TA transversionmutation (13,14). The two activatingmutations, observed in c-Ha-ras genes ofliver tumors induced by treating mice withdiethylnitrosamine (33), were theAT-+TA and A-T->GC that occurredat codon 61.

We also have discovered a new class ofDNA adducts, 3-HA-dU, which are pro-duced by the mutagenic and carcinogenicepoxides ethylene oxide (EO), propyleneoxide (P0), glycidol, epichlorohydrin(ECH), and the epoxide of acrylonitrile(34-38). 3-HA-dU is formed after initialalkylation at the N3 position of dC, fol-lowed by rapid hydrolytic deamination to3-HA-dU (34,37,38). The hydroxyalkylgroup of 3-HA-dU occupies a centralWatson-Crick hydrogen-bonding positionand is likely to disrupt normal base pairing.3-HA-dU is stable in DNA in vitro andmay be the critical promutagenic lesionproduced by aliphatic epoxides in vivo. Therole of 3-HA-dU in mutagenesis byaliphatic epoxides is unknown.

To ascertain the mutagenic potential ofpyrimidine alkylation in DNA, we initiatedin vitro DNA replication studies on Et-dT(39-43) and 3-HA-dU lesions (44,45),site-specifically placed in the same DNAtemplate sequence. In vitro replicationstudies cannot precisely mimic the actualconditions of in vivo DNA synthesis, butthey have proven to be a powerful means tounderstand the mechanisms responsible forthe fine nucleotide selection exhibited byDNA polymerases. The site-modified tem-plate used in the in vitro DNA replicationstudies corresponds to a portion of the bac-teriophage 4X174 genome in the gene Gregion (46). In addition to replicationstudies, the same DNA sequence can beused in separate in vivo site-specific muta-genesis studies, using the 4X174-basedmutagenesis system (25) to facilitate com-parison between in vitro and in vivo muta-genesis studies.

In the presence of the natural metalactivator Mg++, Et-dT and 3-HA-dUadducts interfered with DNA replicationby the Klenow fragment of E. coli DNApolymerase I (Kf Pol I) (39,41,44). Theblock by 04-Et-dT was partial, while o2-Et-dT, N3-Et-dT and 3-HA-dU lesionspresented a complete block to DNAreplication. Since DNA replication past alesion is required to produce mutations, we

1 7-mer Primer 5'-32P-- - -TAAT36-mer Template 3'-OH - --- ATTAGTACAAAT*CTG --- AA5'

I I I17 26 36

Figure 1. DNA replication system for T' = d-Et-dT, d1-Et-dT or N3-Et-dT. For the N3i-Et-dT-containing template, thebase 5' to the lesion is T (3g.

investigated the role of mutagenic metalions, such as Mn++, in mediating DNAsynthesis past Et-dT and 3-HA-dU lesions.Mn++ facilitated mispairing by Et-dT(40,41,43) and 3-HA-dU (44) adductsand subsequent extension of the resultingmispair. Postlesion synthesis implicated Et-dT adducts in AT-*T-A and AT-*G-Cmutations. The epoxide-induced 3-HA-dUlesion may be involved in GC-*A-T tran-sitions. A mechanistic explanation forMn++-mediated mispairing by Et-dT and3-HA-dU is not known. Mn++ binds tonucleotides (47-51) and hence may affecteither template or substrate molecules so asto alter their base-pairing properties.Alternatively, Mn++ may interact withDNA polymerase (50), either reducing theaccuracy of base selection prior to insertion(52) or modifying an exonucleaselikeproofreading function. Mn++ is known tomodify fidelity of DNA replication byDNA polymerases (53-56) and facilitateDNA synthesis past DNA lesions(5,57-59). Mn++, which is a weak muta-gen, has been shown to exert strong co-mutagenic effects with UV (60). In thispaper we summarize the DNA replicationproperties of ethylating agent-induced Et-dT adducts (0 -, 0-- and N3-Et-dT),epoxide-induced 3-hydroxyalkyl-dU [3-HE-dU from EO and 3-hydroxypropy-ldeoxyuridine (3-HP-dU) from P0] andthe role of Mn++ in mediating mutagenesisby these lesions.

Bioloqical Significance ofThymine Ethylation in DNAThe alkylating agent ENU is capable ofinducing a variety of tumor types in abroad range of animal species (10-14) andhumans (9,15). The reactivity of ENUallows it to form a diverse set of DNAadducts both in vitro and in vivo (4,6,16).The order of formation of Et-dT adducts isd-Et-dT > 04-Et-dT > N3-Et-dT. Theselesions are poorly repaired in mammaliansystems (21) and thus may be more biolog-ically important in these systems.Ethylation of dT may alter its base-pairingpattern to form a miscoding lesion such as04-Et-dT (5,61). Alternatively, ethylationmay compromise the ability of the base to

serve as a template during DNA replica-tion, producing noncoding lesions such asN3-Et-dT (39,62) and CY-Et-dT (1,41).Under conditions of relaxed fidelity ofDNA synthesis, the noncoding lesions maymispair to produce mutations.

Transition and transversion mutationsat AT base pairs form an important com-ponent of ethylating agent-induced muta-genesis in SOS-induced bacteria (6,28),human cells (8,29) and animal systems(23), suggesting that dA or dT adductsand/or a breakdown product of theseadducts are responsible for A-T mutations.A*T->G*C transitions can be derived from04-Et-dT by mispairing with dG (27). Acomparison of ENU-induced mutationswith base substitutions produced by otheralkylating agents in bacteria and humancells has led to the suggestion that d-Et-dT may be a significant premutageniclesion in mammalian cells capable ofinducing A*T-4T*A tranversion mutations(8,29,63). Indirect support for this hypoth-esis was derived from mutations observedin vivo in ENU-treated mice, where muta-tions at A-T base pairs accounted for 94%of all mutations (23). Among the A*Tmutations, 55% were A-T-4T-A transver-sions. To ascertain the mutagenic potentialof Et-dT lesions, we studied in vitro DNAreplication properties of each Et-dT lesionsite-specifically incorporated into the sameDNA template. The replication studies uti-lized the primed template system shown inFigure 1.

The replication system contains a 36-nucleotide site-modified DNA templatehybridized to a 32P-labeled 17-nucleotidecomplementary primer. The constructionof site-modified templates has beendescribed (39,42). The Et-dT adducts andtheir derivatives, used in the synthesis ofsite-modified oligomers, were fully charac-terized by thin-layer chromatography, highpressure liquid chromatography (HPLC),ultraviolet, mass and nuclear magnetic res-onance (NMR) spectroscopy. The presenceof the Et-dT moiety in the purifiedoligomer was demonstrated by HPLCanalysis of the nucleosides released fromthe site-modified oligomer following diges-

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Mn+ MEDIATES MUTAGENIC BYPASS OFALKYLPYRIMIDINES IN VITRO

tion with phosphodiesterase and phos-phatase (39,42).

In the DNA replication system (Figure1), the 3'-end of the primer is eightnucleotides away from the thymine modifi-cation (T*) present in the template. Thissystem represents a "running start" forDNA replication in that synthesis occursprior to the polymerase reaching the lesion.The hybridized primer is extended by thepolymerase until T* is encountered. Thefollowing DNA products, reflecting theinfluence of template T*, are feasible. First,the progress of the polymerase is blocked3'to T*. No nucleotide is incorporatedopposite the lesion and a 25-nucleotidepreincorporation blocked product accumu-lates. Second, DNA synthesis terminatesafter incorporating a nucleotide oppositeT*, producing a 26-nucleotide incorpora-tion-dependent blocked product. Finally,DNA synthesis proceeds past the lesionyielding a 36-nucleotide postlesion synthe-sis product. Products of DNA synthesiswere analyzed by polyacrylamide gel elec-trophoresis. Since the 32P-end labeledprimer is used to prime DNA synthesis,each product is only labeled once at the 5'-end. This facilitates the quantitation ofDNA synthesis products in the polymeriza-tion reaction by measuring the radioactiv-ity associated with the individual productbands. The identity of the nucleotideincorporated opposite T* was establishedby DNA sequencing of the 26-nucleotideblocked and the 36-nucleotide postlesionsynthesis products.

DNA Replication Properties of02-Et-dTThe 02-position of dT does not participatein Watson-Crick base pairing. However,ethylation of the 02-position of dT inter-feres with normal hydrogen bonding of dTwith dA, by fixing the thymine base in theenol tautomer with the loss of a hydrogenatom at the central hydrogen-bonding site(N3) of dT (42). Disruption of normalbase pairing may inhibit DNA synthesis.This is consistent with our DNA replica-tion studies where, in the presence of thenatural metal activator Mg", 02-Et-dTblocked DNA replication by Kf Pol I pre-dominantly 3' to the lesion (41). DNAsynthesis past the lesion was negligible(<1%). Incorporation of dA opposite o-Et-dT occurred with increasing deoxyri-bonucleoside-5'-triphosphate (dNTP)concentrations (41), which was furtherenhanced by inhibiting the 3'-5' exonu-clease proofreading activity of the poly-merase with deoxyadenosine-5'-phosphate.

The postlesion synthesis remained negligi-ble (41). The 0--Et-dT'dA base pair mayoccur with the formation of two hydrogenbonds between the 04 of O-Et-dT and theN6 hydrogen atom of dA, and between theN3 of (I-Et-dT and the protonated M ofdA. A similar hydrogen bonding schemehas been suggested for 04-Et-dT-dA byNMR studies (64). The (I-Et-dT.dA basepair with two hydrogen bonds is expectedto be thermodynamically stable. This isconsistent with thermal denaturing studieswhere (I-Et-dT, present in the alternatingpoly[d(A.T)]polymers, did not alter thethermal melting profile (65).

Inhibition of DNA synthesis afterincorporation of dATP opposite template0(-Et-dT (41) or 02-Et-dT-5'-triphos-phate opposite template dA (66) suggeststhat the geometric conformation of the o2-Et-dT-dA base pair deviates significantlyfrom that of the normal Watson-Crick pairand may adopt a wobble conformation(64,67). In the wobble conformation,phosphodiester links (both 3' and 5' to dA)may have to be distorted to accommodatethe 0 -Et-dT-dA base pair in a DNAhelix. This hypothesis is consistent withmolecular and computer modeling studies,indicating that the presence of (I-alkyl-dTin DNA may cause distortion in the DNAstructure (1). Additional support for thishypothesis is derived from 31P NMR stud-ies of DNA duplexes, containing the wob-ble base pairs 06-Et-dG dC and04-Et-dT-dA (64). Distortion of thephosphodiester links 3' and 5' to dT anddG, respectively, was observed. The con-formational changes associated with phos-phodiester bonds during the formation ofthe (I-Et-dT.dA base pair are expected toadversely effect the catalysis of phosphodi-ester links on both the 3' and 5' sides of theincoming dA opposite 02-Et-dT duringDNA replication. This would suggest thatincorporation of dA opposite O-Et-dTand extension of the resulting 02-Et-dT-dA base pair will be inefficient. This issupported by in vitro DNA replicationstudies in the presence of Mg", whereDNA synthesis was terminated predomi-nantly (94%) 3' to 0-Et-dT, and postle-sion synthesis did not occur (41). Similarresults were obtained during DNA replica-tion using bacteriophage T7 DNA poly-merase (T7 Pol) (42). The block by the02-Et-dT.dA base pair appears to be aninherent property of the spatial conforma-tion of this base pair, since it was observedrepeatedly during synthesis by Kf Pol l (41)or T7 Pol (42) in the presence of Mg++ orMn++ from running (41,42) or standing

(68) starts. Under normal cellular condi-tions, extension of the (I-Et-dT.dA basepair may either not occur or may occurwith low efficiency. Our DNA replicationstudies in the presence of Mg++ (41) sug-gest that (I-Et-dT may contribute in partto the cytotoxicity of ethylating agents.

Since DNA replication past the lesionis a necessary step in the production ofmutations, we investigated the role of themutagenic metal ion Mn++ in mediatingDNA synthesis past the (I-Et-dT adduct.When Mn++ was substituted for Mg++ inthe polymerization reaction, incorporationof a nucleotide opposite 02-Et-dT andsubsequent postlesion synthesis wereenhanced (41). Increasing the dNTP con-centration and inhibiting the proofreadingactivity of Kf Pol I increased postlesionsynthesis (which reached 66% at 200 pMdNTP) (41). DNA sequencing of theblocked and postlesion synthesis productsrevealed that while dA was present oppo-site 02-Et-dT in the blocked product,both dA and dT were present opposite thelesion in the postlesion synthesis product(41). The presence of dA opposite (I-Et-dT in both the blocked and postlesionsynthesis products indicates that, in thepresence of Mn+, the 0-Et-dT.dA basepair at the 3'-end of the growing chaincan be extended but inefficiently. This isin contrast to DNA replication in thepresence of Mg", where the 02-Et-dT-dA base pair was not extended.Absence of dT opposite (I-Et-dT in theblocked product suggests that formationof an (&-Et-dT.dT base pair at the repli-cation fork is efficiently extended. Theresults implicate (I-Et-dT in transversionmutagenesis at A-T base pairs and suggesta role for Mn++ in mediating AT-+TAtransversion mutation by (I-Et-dT.

Formation of a pyrimidine-pyrimidinebase pair is rare. The O-Et-dT.dT basepair probably has one hydrogen bondforming between the N3 nitrogen atoms.The pairing of two pyrimidines wouldallow a long hydrogen bond, which woulddecrease steric hindrance between theethyl group of (I-Et-dT and the carboxylgroup at C2 of dT. This hydrogen-bond-ing scheme could result in a normal sugar-phosphate backbone with the 02-Et-dTbase pair retaining the Watson-Crickalignment. The normal Watson-Crickalignment of the (I-Et-dT.dT mispairwould facilitate formation of phosphodi-ester bonds on both the 3' and 5' sides ofdT. This is consistent with our DNAreplication studies (41), where incorpora-tion of dT opposite (I-Et-dT was effi-

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BHANOTAND SOLOMON

ciently extended. Other mis4pairs, includ-ing 06-Et-dG-dT and 0 -Et-dT-dG,which contain one hydrogen bond andretain normal Watson-Crick alignment,have been shown to be efficientlyextended in vitro (61,69). These studiessuggest that correct alignment of thebackbone is crucial in DNA replication(64,67). The strength of hydrogen bond-ing is of secondary importance. Molecularand computer models together withphysicochemical studies on the 02-Et-dT-dT base pair will provide insight intothis hypothesis.

The kinetic mechanisms by which oEt-dT impedes DNA synthesis and mis-codes were studied (70). The kineticparameters, K. and Vmax, for dA and dTinsertion opposite and extension past o-Et-dT by Kf Pol I in the presence of Mg++and Mg", were determined using a poly-acrylamide gel assay (71-73). Insertionand extension frequencies of 02_-Et-dT.dA and d-Et-dT.dT base pairs, rela-tive to the right base pair (dT.dA), wereestimated from V1mnIK ratios. The pre-ma mliminary results revealed (70) that, in thepresence of Mn+, 02-Et-dT inhibitedinsertion and extension of dA and dT atthis lesion with efficiencies of 10-4 orlower. As compared to Mg++, Mn++increased insertion frequencies of dA anddT opposite 02-Et-dT by 10-fold ormore. The insertion was enhanced pri-marily through Km discrimination. Theextension frequency at the 02-Et-dT.dTbase pair was enhanced 6-fold when Mn++was substituted for Mg++ in the polymer-ization reaction. Mn++ had little effect onextension of the 02-Et-dT-dA base air.As compared to d-Et-dT-dA, the 0-Et-dT.dT mispair was extended 30 timesmore efficiently (70). A higher extensionfrequency of the 02-Et-dT-dT mispairwas primarily achieved through anincrease in V.max The results suggest that,as compared to Mg++, Mn++ may increasethe residence time of the 02-Et-dT-dTmispair at the catalytic site of the poly-merase. These kinetic studies are consis-tent with in vitro DNA replicationstudies of 0 -Et-dT discussed above(41,42). They suggest that the 02-Et-dT.dA base pair plays a central role inthe inhibition of DNA synthesis by 02-Et-dT and is consistent with the notionthat this base pair may cause distortionin the DNA structure (1). Efficient inser-tion and extension of dT at 02-Et-dTsuggest a role for Mn++ in mediatingA-T-4T-A transversion mutagenesis byci-alkyl-dT lesions.

DNA Replication Properties of04-alkyI-dTThe established role of 06-alkyl-dG in themutagenesis (2,25,26) and initiation ofcarcinogenesis (12,17) by alkylatingagents, obscured the possible similar roleof other alkyl DNA adducts. Recently,increased attention has been focused onO-alkylpyrimidines. Several studies havecorrelated the persistence of 04-alkyl-dTin target tissue with organ specificity oftumors resulting from N-nitrosoalkylatinygagents (18-20). 5'-Triphosphates of 0 -alkyl-dT were able to substitute for dTTPin poly[d(A.T)] synthesis by E. coli DNApolymerase (65,74). The resulting poly-mer, poly[d(A-T, 04-alkyl-dT)] supportedthe incorporation of dG during in vitroDNA replication by the same polymerase.The results suggest the likely pairing of04-alkyl-dT with dG as well as with dA.The role of 04-alkyl-dT in A-T-*G-Ctransition was unambiguously establishedthrough site-specific mutagenesis studieson 04-Me-dT (27). The alkyl group of04-alkyl-dT is located within the Watson-Crick base pairing region and may inter-fere with normal hydrogen bonding of dTwith dA. The 04-alkyl-dT lesion maybehave as a miscoding and/or noncodinglesion during DNA replication. The non-coding lesions usually inhibit DNA repli-cation by distorting the DNA structure.Miscoding lesions alter the precision ofbase pairing during DNA synthesis lead-ing to mutation. The role of 04-alkyl-dTin blocking DNA synthesis and the reac-tion conditions allowing DNA synthesispast the lesion were studied through invitro DNA replication studies of 0 -Et-dT, site-specifically incorporated into aDNA template. The replication studiesutilized the primed-template shown inFigure 1.

In the presence of the natural metalactivator Mg", 04-Et-dT presented a par-tial (54%) block to DNA replication byKf Pol I, with replication mainly (48%)interrupted 3' to 04-Et-dT. DNA synthe-sis past the 04-Et-dT lesion was 46%.Accumulation of the blocked product,obtained after incorporation of anucleotide opposite 04-Et-dT, was low(6%) and remained constant over a widerange of dNTP concentrations (10-200pM). The results suggest that, duringDNA replication past 04-Et-dT in thepresence of Mg", insertion of anucleotide opposite this lesion may be therate-limiting step. The replication blockby 04-Et-dT was removed when Mn++was substituted for Mg++ and a postlesion

synthesis product was obtained in highyield (>90%). DNA sequencing revealedthat predominantly dG was incorporatedopposite 04-Et-dT (Figure 2) duringDNA synthesis past the lesion. The resultsimplicate Mn++ in facilitating the inser-tion of dG opposite 0 -Et-dT and exten-sion of the resulting base pair.

Kinetics of insertion opposite template0 -Me-dT have been described (61).From Vmax/Km ratios, the pairing of 04-Me-dT-dG was preferred 10-fold overthat of 04-Me-dT-dA. The relative incor-poration efficiencies of dA and dG oppo-site 04-Me-dT appears to reflect therelative rates of base pair formation

C C G GA

G

Figure 2 DNA sequence analysis of the postlesion syn-thesis product synthesized on an L1-Et-dT-containing tem-plate by Kf Pol in the presence of Mn^. A Maxam-Gilbertsequencing gel is shown. The presence of bands in dG-specific lanes at position 26 indicates incorporation of dGopposite 04-Et-dT during postlesion synthesis.

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Mn"+ MEDIATES MUTAGENIC BYPASS OFALKYLPYRIMIDINES IN VITRO

(nucleotide insertion) rather than theeffect of proofreading because the efficien-cies using Kf Pol I were similar to thoseusing the Drosophila melanogaster poly-merase a-primase complex, which doesnot contain detectable 3'-*5' exonucleaseproofreading activity (75). In the absenceof any dNTP, the "weak" 3'-5' exonu-clease activity of Kf Pol I did not excisethe 04-Me-dT'dG base pair (5). This isconsistent with our DNA replication stud-ies where the 04-Et-dT'dG base pair canbe easily extended.NMR studies on DNA duplexes con-

taining 04-Me-dT have indicated that the04-Me-dT-dA base pair has a wobbleconformation with the alkylated basemoved towards the major groove of thehelix (64). A wobble alignment for 04-Me-dT-dG was ruled out and it was sug-gested that this base pair retains thenormal Watson-Crick alignment (64).Due to steric hindrance by the 04-methylgroup, the normal alignment may haveonly one hydrogen-bond between the 2-amino of dG and the o2 of 04-Me-dT.The 04-Me-dT-dG mispair having lesshydrogen bonding than 04-Me-dT-dA isconsistent with the optical melting pro-files of duplexes where the duplex con-taining 0 -Me-dT'dA pairs melted athigher temperatures than the one contain-ing 04-Me-dT-dG pairs (76).

An important factor in miscoding by04-Me-dT is that the 04-Me-dT.dG mis-pair retains the Watson-Crick alignment,with no distortion of phosphodiester links3' and 5' to the dG, as revealed by NMRstudies (64). This is consistent with thegreater efficiency of dG incorporationopposite 04-Me-dT as compared to dA(61). Stimulation of dG incorporationopposite 04-Et-dT in the presence ofMn+ in our studies suggests a role forMn++ in stabilizing the 04-Et-dT'dGmispair in a normal Watson-Crick align-ment to facilitate the formation of phos-phodiester bonds. Based on NMR studieson nucleotide binding to E. coli DNApolymerase I (77), it has been suggestedthat the polymerase-Mn++ complex maybe less selective of the sugar ring confor-mation of the nucleotide than the enzymecomplexed with Mg". Binding of thedNTP substrate to the active site of thepolymerase in the presence of Mn++ mayoccur in conformations favorable forWatson-Crick base pairing in DNA Bform. These types of conformations maynot be favored in the presence of Mg".The DNA replication studies suggest arole for Mn++ in stimulating A-TG-C

transition mutagenesis by 04-alkyl-dTlesions.

DNA Relication Properties ofN3-E -dN3-alkyl-dT is formed, both in vitro andin vivo, but in relatively small amounts(4,6,16). Among the alkylating agents,ENU demonstrates a significant amount ofbinding to O-alkyl and N3-alkyl pyrim-idines. N3-alkyl-dT is stable in DNA invitro. No DNA repair activity has beenreported for N3-alkyl-dT. This DNAlesion may be persistent in vivo and exertsits biological consequences long after expo-sure has occurred. The alkyl group of N3-alkyl-dT occupies the central Watson-Crick hydrogen bonding site (N3) ofthymine and is likely to interfere with nor-mal hydrogen bonding of dT, probablyleading to mispairing and/or inhibition ofDNA synthesis. N3-alkyl-dT is likely to bea potentially cytotoxic and mutageniclesion produced by alkylating agents. Thebiological significance of the N3-alkyl-dTlesion was ascertained through in vitroDNA replication studies of N3-Et-dT(39,40,43) present at a single site in theDNA template shown in Figure 1.

In the presence of the natural metalactivator Mg++ and a low dNTP concen-tration (10 11M), N3-Et-dT blocked DNAsynthesis by Kf Pol I predominantly 3' toN3-Et-dT. DNA synthesis past the lesionwas not observed (39). Incorporation of dAopposite N3-Et-dT occurred with increas-ing dNTP concentrations, but no postle-sion synthesis was obtained (39). Similarresults were obtained during DNA replica-tion with T7 Pol, where incorporation ofdA opposite N3-Et-dT blocked DNA syn-thesis in the presence of Mg++ (43). Thesestudies implicate N3-Et-dT as a potentiallycytotoxic lesion produced by ethylatingagents.

No postlesion synthesis suggests thatthe N3-Et-dTdA base pair, formed at thereplication fork, did not retain the Watson-Crick alignment and may cause distortionin the DNA structure. Due to steric hin-drance by the lesion, translocation of thepolymerase to the nucleotide past thelesion may be extremely slow. Polymer-ization of the nucleotide past N3-Et-dTmay also be very slow, owing to the need toextend the distorted terminus formed bythe wobble N3-Et-dT-dA base pair.During a pause at the lesion, the poly-merase may dissociate from the lesion-blocked primer-template complex andcease elongation. Once dissociated, rebind-ing of the polymerase to the primer-tem-

plate complex may lead to formation of adefective initiation complex of lower stabil-ity. This complex may be stable enough toallow the relatively easy first polymerizationstep of inserting dA opposite N3-Et-dT,but not the next very slow step of extend-ing the N3-Et-dT*dA base pair. Thisnotion is consistent with DNA replicationstudies (39), where in the presence of Mg++the blocked product, obtained after incor-poration of dA opposite N3-Et-dT, wasformed but not extended.

When Mn++ was substituted for Mg",incorporation of dA opposite N3-Et-dTwas increased from 4% in the presence ofMg++ to 60% in the presence of Mn++ at10 ,uM dNTP (40). At this dNTP concen-tration, DNA synthesis past the lesion wasnot obtained. Postlesion synthesis occurredat higher dNTP concentrations andreached 68% at 200 pM. During postle-sion synthesis, dT was incorporated oppo-site N3-Et-dT (40), implicating this lesionin transversion mutagenesis at the A-T basepair by ethylating agents. The results sug-gest a role for Mn++ in mediating anAT--TA transversion mutation by theN3-Et-dT lesion.

The absence of dT opposite N3-Et-dTin the blocked product and its presenceonly in the postlesion synthesis product(40) suggest that the N3-Et-dT-dT mis-pair formed at the replication fork is notinhibitory to DNA synthesis. This mispairis efficiently extended, leading to anAT-*TA mutation. These results aresimilar to those observed in the case of (I-Me-dG-dT (64), 04-Me-dT-dG (64) and(Y-Et-dT-dT (41,42) mispairs, which wereefficiently extended and led to mutations.As shown for d-Me-dG.dT and 04-Me-dT-dT mispairs (64), the N3-Et-dT-dTmispair may also retain the normalWatson-Crick alignment, facilitatingextension of this mispair. This is consistentwith postlesion synthesis in high yield(68%) observed in our DNA replicationstudies (40).

Since the N3-Et-dT'dT mispair is effi-ciently extended, incorporation of dTopposite N3-Et-dT appears to be the rate-limiting step during posdesion synthesis. Incontrast to the low dNTP concentration(10 pM), higher dNTP concentrationsfacilitated insertion of dT opposite N3-Et-dT (40). Polymerization of the next correctnucleotide following the N3-Et-dT-dTmispair protected the mispair from excisionby the proofreading activity of the poly-merase. Since the polymerase has difficultyextending from the N3-Et-dT-dA basepair, this base pair becomes susceptible to

Volume 102, Supplement 3, September 1994 85

BHANOTAND SOLOMON

12-mer Primer 5'-32P---- -TMT50-mer Template 3'-OH ---- ATTAGTACAAAAU*CTGAMA --- TCGA-5'

I I I12 21 50

Figure 3. DNA replication system for U* = 3-HE-dU or 3-HP-dU.

proofreading. Excision of dA from N3-Et-dT-dA makes the template N3-Et-dTlesion available for insertion of dA and dTopposite the lesion with dT insertion effi-ciently extended. The net result of thiscycling is accumulation of the postlesionsynthesis product, containing dT oppositeN3-Et-dT in high yield (68%) (40).

In contrast to Kf Pol I, T7 Pol washighly specific in incorporating only dTopposite N3-Et-dT in the presence ofMn++ during postlesion synthesis (>95%)even at 10 pM dNTP (43). The greaterability of T7 Pol to incorporate dT oppo-site N3-Et-dT may reflect the large differ-ence in processivity between T7 Pol and KfPol I (67). T7 Pol incorporates thousandsof nucleotides prior to dissociating fromthe template (78). The high affinity of T7Pol for the DNA template may increase thelikelihood of insertion and extension of dTopposite N3-Et-dT by the polymerase.The high yield of postlesion synthesis prob-ably was achieved through protection ofthe N3-Et-dTdT mispair by extensionand excision of the N3-Et-dTdA base pairby the highly potent 3'-*5' exonucleaseproofreading activity associated with T7Pol.

Biological Significance ofAliphatic Epoxide-induced3-HA-dU LesionsAliphatic epoxides include a number ofvery reactive reagents. They contain three-membered rings, which are highly strained.The least substituted carbon, which is steri-cally more accessible, is the site of attack ofmost epoxides (SN2 mechanism) underneutral conditions. These epoxides are aclass of direct-acting alkylating agents,which are effective mutagens and animalcarcinogens (79-81). Because of their use-fulness as chemical reagents, the simplevolatile epoxides, especially EO, PO, andECH, are used extensively in industry andresult in potential human exposure in thework place. An association between expo-sure to EO and cancer has led to the sug-gestion that EO may be involved in theetiology of human cancer. PO and ECHhave been shown to be rodent carcinogens.

Alkylation of DNA, followed by thepersistence of premutagenic adducts duringthe period of DNA replication and cell

division, seem to be necessary but not suffi-cient requirements for initiation of carcino-genesis with many alkylating agents. Wediscovered a new class of DNA adducts(34-37), 3-HA-dU, which is produced bymutagenic and carcinogenic epoxides. The3-HA-dU lesion is stable in DNA in vitro.This lesion is formed in DNA after rapidhydrolytic deamination of a 3-hydrox-yalkyldeoxycytidine (3-HA-dC) adduct(34,38). The initial reaction of SN2 epox-ides occurs at N3 of dC. The charge on theprotonated 3-HA-dC intermediate (pK=9)is delocalized over the C4 carbon, which isthen attacked by the side chain OH group,resulting in the loss of ammonia (deamina-tion) with retention of the charge at C4.Hydroxyl attack at this carbon results inring opening and formation of 3-HA-dU(Solomon, unpublished). The halflives ofhydrolytic deamination of 3-HE-dU and3-HP-dU at pH 7.4 and 37°C were 10 hrand 6 hr respectively (34,37).

The 3-hydroxyalkyl group of 3-HA-dUoccupies a central Watson-Crick hydrogenbonding position and is likely to disruptnormal hydrogen bonding, leading to mis-pairing and/or inhibition of DNA replica-tion. 3-HA-dU may be the criticalpremutagenic lesion produced by SN2epoxides in vivo. In order to understandthe role of 3-HA-dU in mutagenesis andcancer induction, we initiated in vitroDNA replication studies of 3-HE-dU and3-HP-dU, produced by EO and POrespectively.

The replication system for 3-HA-dU(Figure 3) is similar to the one used for Et-dT adducts (Figure 1), except that theprimer used was a 12-mer and the site-modified DNA template was a 50-mer.Use of the 50-nucleotide template wasnecessitated by the need to facilitatesequencing of the postlesion synthesisproduct by Sanger's dideoxy-sequencingprotocol, and to incorporate the site-modi-fied 50-mer into a 50-nucleotide gap in~X174 replicative form DNA for in vivosite-specific mutagenesis studies. To facili-tate comparison of the results between Et-dT and 3-HA-dU adducts, U* was locatedin the same DNA sequence from 4X174.The site-modified oligomers were fullycharacterized for their purity, expectedDNA sequence and presence of U* in

essentially all oligomer molecules. Thedetails of the synthesis of site-modifiedoligomers and their characterization will bedescribed elsewhere. In this replication sys-tem, the expected DNA synthesis productsare a 20-nucleotide preincorporation-blocked, a 21-nucleotide incorporation-blocked and a 50-nucleotide postlesionsynthesis product.

DNA Replication Properties of3-HE-dUAs observed with the analogous mutageniclesion, N3-Et-dT, 3-HE-dU blocked DNAsynthesis by Kf Pol I (Figure 4). In thepresence of the natural metal activatorMg++ and at 10 pM dNTP, the majority(70%) of the block was 3' to 3-HE-dU andthe remainder (27%) was blocked afterincorporating a nucleotide opposite 3-HE-dU (44). Synthesis past the lesion was neg-ligible (<3%). Incorporation opposite3-HE-dU increased with increasing dNTPconcentrations, reaching 60% at 200 pM.Postlesion synthesis remained negligible(<3%). DNA sequencing of the 21-nucleotide blocked product revealed that

w w .

10pM 4NTP 200pM CdNTP

Figure 4. In vitro DNA replication catalyzed by Kf Pol inthe presence of Mg+ on a template containing 3-HE-dUand 3-HP-dU at a single site.

5o -

20 -

12 -

Environmental Health Perspectives86

Mn*+ MEDIATES MUTAGENIC BYPASS OFALKYLPYRIMIDINES IN VITRO

A-

G G C CA T

Figure 5. DNA sequence analysis of the 21-nucleotideblocked product synthesized on a 3-HE-dU-containing tem-plate by Kf Pol in the presence of Mg++. A Maxam-Gilbert sequencing gel is shown. Presence of a band in thedA-specific lane at position 21 indicates incorporation ofdA opposite 3-HE-dU during the synthesis block.

dA is incorporated opposite 3-HE-dU(Figure 5). Since postlesion synthesis isnegligible, the results suggest that the 3-HE-dU-dA present at the growing replica-tion fork is inhibitory to DNA synthesis.The results are similar to the analogousN3-Et-dT lesion, where the N3-Et-dT-dAbase pair was not extended in the presenceof Mg++ (39). Our DNA replication studiesimplicate 3-HE-dU as a potentially-cyto-toxic lesion produced by EO.

During in vitro DNA replication, the3-HE-dU-dA base pair behaved in a man-ner similar to N3-Et-dT-dA, 02-Et-dT-dA, 04-Et-dT-dA, and d5-Me-dG.dCbase pairs. As suggested for the 04-Me-dT-dA and d-Me-dG.dC base pairs (64),the 3-HE-dU-dA base pair may also existin a wobble conformation and cause distor-

tion in the DNA structure, making exten-sion of this pair difficult. This increases theexposure time of the 3-HE-dU-dA basepair to the 3'->5' exonuclease activity ofthe polymerase, making the base pair sus-ceptible to excision by proofreading. Thiswas manifested in our DNA replicationstudies when Kf Pol I (exo-) (deficient in3'-<5' exonuclease) was substituted for KfPol I (exo+). As expected, post lesion syn-thesis was dramataically increased from<3% for Kf Pol I to <50% for Kf Pol I(exo-).

Substitution of Mn++ for Mg++increased incorporation opposite 3-HE-dUand subsequent synthesis past the lesion(Figure 6). At 200 pM dNTP, postlesionsynthesis increased from <3% in the pres-ence of Mg++ to >85% in the presence ofMn++. The specificity of nucleotide incor-poration opposite 3-HE-dU is under inves-tigation.

The Mn++-mediated synthesis past 3-HE-dU suggests that the role of Mn++ inmodifying the fidelity of Kf Pol I in DNAreplication may be comparable to the SOS-induced functions in bacteria. In bacteria,SOS-induced proteins may alter the fidelityof the DNA replication complex, facilitat-ing the incorporation and subsequentextension at 3-HE-dU. This hypothesissuggests that mutagenesis by aliphaticepoxide-induced 3-HA-dU requires induc-tion of the SOS system in bacteria. This issupported by the production of SOS-dependent mutagenesis by PO at templatecytosines (45). Involvement of the SOS-like system in mammalian cells is notknown. Inside the mammalian cell, DNApolymerase-accessory proteins may facili-tate incorporation and subsequent exten-sion at 3-HA-dU. Since 3-HA-dU isderived from deamination of aliphaticepoxide-induced 3-HA-dC, extension of allbase pairs (except 3-HA-dU.dG) at 3-HA-dU will produce mutations. Our in vitroDNA replication studies have demon-strated formation of a 3-HE-dU-dA basepair. In vivo extension of this pair will pro-duce a GC->A-T transition mutation andimplicate the 3-HE-dU lesion inG-C-4AT transition mutagenesis by EO.Support for this hypothesis is derived fromour mutagenesis studies with PO, whereG-C-A-T transitions represent an impor-tant component of PO-induced mutationalspectra (45).

DNA Replication Properties of3-HPdUPO-induced 3-HP-dU behaved in a similarmanner as 3-HE-dU (44) and the analo-

gous N3-Et-dT lesion during in vitro DNAreplication (39,40). 3-HP-dU blockedDNA synthesis by Kf Pol I in the presenceof Mg++ 3' to 3-HP-dU and after incorpo-rating a nucleotide opposite the lesion(Figure 4). Postlesion synthesis was negligi-ble. Substitution of Mn++ for Mg++ medi-ated DNA synthesis past 3-HP-dU. Thespecificity of the nucleotide incorporatedopposite 3-HP-dU in the blocked andpostlesion synthesis products is beinginvestigated.

Role of Mn" in MediatingMispairingManganese is known to be highly muta-genic in vivo and to reduce the fidelity ofDNA synthesis in vitro (82). It also showsa strong co-mutagenic effect with UV (60).The mechanism of Mn++-induced mutage-nesis has been studied using E. coli DNApolymerase I (50). The role of a free Mn++concentration on Mn++-induced mutagene-

Figure 6. In vitro DNA replication catalyzed by Kf Pol inthe presence of Mnw on a template containing 3-HE-dU ata single site.

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BHANOTAND SOLOMON

sis in vitro was determined by an analysis ofthe polymerase error rate and a comparisonwith dissociation constants of Mn++ fromthe enzyme, template, and dNTP. Thesestudies suggest that, in the presence ofphysiologically-relevant free Mn++ concen-trations (10-100 pM), Mn++ induced mis-incorporations by interacting with theDNA template rather than with the poly-merase (50). At higher Mn++ concentra-tions (0.5-1.5 mM), Mn++-inducedmutagenesis is probably due to Mn++association either with single-strandedregions of template DNA or with weaksites in the polymerase. The results with T4DNA polymerase suggest that the muta-genic action of Mn++ can be attributed pri-marily to a significant differential increasein binding of mispaired relative to correctlypaired nucleotides to the polymerase-tem-plate complex (49). The resulting increasein residence times for mispairednucleotides on the complex results in theirincreased frequency of misinsertion. Asmaller contributing factor to Mn++-induced mutagenesis was the loss of proof-reading specificity (49). These conclusionsare supported by other kinetic studies ofincorrect vs. correct incorporation duringDNA synthesis (51,83).

The alkyl group of alkylated pyrim-idines studied by us, whether located at aWatson-Crick base-pairing position (i.e.,N3-Et-dT, 3-HE-dU, 3-HP-dU and 04-Et-dT) or not (i.e., d-Et-dT), interfereswith normal hydrogen bonding of thealkylpyrimidines, leading to mispairing orblocking of DNA synthesis. Since normalhydrogen bonding is disrupted, anucleotide inserted opposite alkylpyrimi-dine represents a mismatch and may berecognized by the 3'->5'-exonucleaseproofreading activity of the polymerase.Mispairs that retain the Watson-Crickalignment are efficiently extended, leadin,gto misincorporation. Mispairs, such as (I-Me-dG-dT and 04-Me-dT-dG, shown toretain Watson-Crick conformation, are

efficiently extended (64). Mispairs thatadopt a wobble conformation are either notextended or extended inefficiently, owingto the distortion caused by the mispair inthe DNA structure. We postulate that therole of Mn++ in mediating mispairing byalkylpyrimidines is to increase the probabil-ity of inserting a nucleotide opposite thelesion in a conformation that retains theWatson-Crick alignment. In our DNAreplication studies, a higher Mn++ concen-tration (500 jiM) was used. At this concen-tration, Mn++ binds to DNA polymerase,template DNA, and dNTP substrates (50).The normal Watson-Crick alignment ofthe mispairs formed by the 0O-,0 andN3-Et-dT lesions may have been achievedthrough interactions of Mn++ with thepolymerase-template-dNTP complex.NMR studies of nucleotide binding to E.coli DNA polymerase I have shown thatbinding of the dNTP substrate to theactive site of the polymerase in the presenceof Mn++ may occur in conformations favor-able for Watson-Crick base pairing in DNAB form. These types of conformations maynot be favored in the presence of Mg++(77). This hypothesis is consistent with ourDNA replication studies, where 02-Et-dT-dT, O4-Et-dT-dG and N3-Et-dT*dTmispairs are formed and efficiendy extendedin the presence ofMn++ but not Mg".ConclusionAlkylation of thymine in DNA is toxic,mutagenic, and carcinogenic. Et-dTadducts block in vitro DNA synthesis,often after incorporating dA opposite thelesions. In vivo extension of the Et-dT-dAbase pair is nonmutagenic. Failure toextend the Et-dT'dA base pair implicatesEt-dT lesions in cytotoxicity by ethylatingagents. Mn++-mediated mispairing andbypass of Et-dT adducts suggest a co-mutagenic role for Mn++ in the mutagenic-ity of ethylating agents and implicatesEt-dT adducts in A T-4T-A andA-T-*G*C mutations.

The implication, that ENU-inducedEt-dT lesions can produce A-ToTA andAT->GC mutations, and the observationof these mutations at the activating site ofoncogenes isolated from ENU-inducedtumors, emphasize the existence of animportant, but unproved, relationshipamong the formation of N3-Et-dT, d-Et-dT and 04-Et-dT lesions, the induction oftransversion and transition mutations atA*T base pairs, and the subsequent devel-opment of cancer by N-nitrosoethylatingagents. The prevalence of N-nitroso com-pounds in the environment and their for-mation in vivo have led to the suggestionthat they may be involved in the etiology ofhuman cancers (9). The demonstration ofN-nitroso alkylating agent-induced muta-tions in activated oncogenes (11,13,14,32suggests that cellular protooncogenes repre-sent important targets for these agents.Although Et-dT adducts are rapidlyrepaired in bacteria (30), their repair inmammalian systems is not efficient (21).The Et-dT adducts are among the highly-persistent DNA ethylation products inmammalian systems (19,22. Our in vitroDNA replication studies suggest that ethyl-ation of thymine in DNA is biologicallysignificant and may exert cytotoxic, muta-genic and carcinogenic activity long afterthe exposure has occurred.

Preliminary DNA replication studiesof 3-HE-dU and 3-HP-dU suggest a dualrole for epoxide-induced 3-HA-dUlesions. They block DNA replication invitro and may terminate DNA synthesisin vivo, contributing to the cytotoxicity ofaliphatic epoxides. Under relaxed poly-merase fidelity (Mn++), DNA synthesispast 3-HE-dU and 3-HP-dU occurs,implicating these lesions in mutagenesis atGC base pairs by epoxides. The studiesprovide a basis for understanding molecu-lar mechanisms by which environmentallyimportant aliphatic epoxides producemutations and contribute to the processof carcinogenesis.

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