Effect of site-specifically located aristolochic acid DNA adducts on in vitro DNA synthesis by human DNA polymerase α

ELSEVIER

Cancer Letters 98 (1995) 47-56

CANCER LETTERS

Effect of site-specifically located aristolochic acid DNA adducts on in vitro DNA synthesis by human DNA polymerase a Thomas H. Broschard 1, Manfred Wiessler, Heinz H. Schmeiser* Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

Received 21 August 1995; accepted 20 September 1995

Abstract

In order to examine the effect of purine adducts of the plant carcinogen aristolochic acid (AA) on DNA replication four 30-mer templates were prepared which contained single site-specific AA lesions. The oligonucleotides were isolated by HPLC and shown to contain the two known aristolochic acid I-DNA adducts (dA-AAI, dG-AAI) or the two known aristolochic acid II-DNA adducts (dA-AAII, dG-AAII) at position 27 from the 3' end by 32p-postlabeling. These adducted templates were replicated in primer (23-mer) extension reactions catalysed by human DNA polymerase a. Both AAI-DNA adducts (dA-AAI, dG-AAI) blocked DNA synthesis predominantly (80-95%) at the nucleotide 3' to the adduct, although primer extension to the full length of the template was found with unmodified control templates. Increasing dNTP concentrations had only a small effect on the DNA synthesis and translesional synthesis was negligible. In contrast, both AAII-DNA adducts showed marked differences in primer extension reactions. Blocking of DNA synthesis by the dA-AAII adduct was strongly dNTP dependent. With increasing dNTP concentrations 27 and 28 nucleotide products, indicating termination of DNA synthesis after incorporation of a nucleotide opposite this adduct and incorporation of an additional nucleotide accumulated. Only the dG-AAII adducted template allowed substantial translesional synthesis to the full length of the template (up to 25%). When a 26-mer primer was used to examine nucleotide incorporation directly across from the four purine adducts, we found no detectable incorporation of nucleotides for the dA-AAI adduct, whereas the dG-AAI adduct and both AAII-adducts (dA-AAII and dG-AAII) allowed preferential incorporation of the correct nucleotide. These results indicate that for human polymerase a three AA purine adducts (dA-AAI, dG-AAI and dA-AAII) provide severe blocks to DNA replication and that dG-AAII, which allows translesional synthesis, may not be a very efficient mutagenic lesion. Keywords: Aristolochic acid; DNA adducts; In vitro replication; DNA polymerase a; Primer extension reactions

Abbreviations: AA, aristolochic acid; AAI, adstolochic acid I (8methoxy-6-nitrophenanthro[3,4-d]-1,3-dioxolo-5-carboxylic acid; AAII, aristolochic acid II (6-nitrophenanthro[3,4-d]-l,3-dioxolo5-carboxylic acid; dA-AAI, 7-(deoxyadenosin-N6-yl)-aristolactam I; dG-AAI, 7-(deoxyguanosin-N2-yl)-aristolactam I; dA-AAII, 7(deox~adenosin-N6-yl)-aristolactam II; dG-AAII, 7-(deoxyguanosin-N~-yl)-adstolactam II; TEAA, triethylammonium acetate; pol a, human DNA polymerase a.

* Corresponding author. Tel.: +49 6221 423348; Fax: +49 6221 423375. I Present address: UPR Canc6rogen~se et Mutagen~se Mol6culaire et Structurale, CNRS, ESBS, Boulevard Sebastien Brant, 67400 lllkirch, France.

0304-3835/95/$09.50 9 1995 Elsevier Science Ireland Ltd. All fights reserved SSDI 0304-3835(95)04010-2

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T.H. Broschard et al. / Cancer Letters 98 (1995) 47-56

to

1. I n t r o d u c t i o n

Some of the most potent carcinogens known, such as safrole, estragole, cycasin and the group of pyrrolizidine alkaloids are of plant origin [1]. In 1982, Mengs and co-workers [2] reported aristolochic acid

(AA), a plant extract of A r i s t o l o c h i a species, be a potent forestomach carcinogen in rats. Prior to 1982, A A was used as an antiinflammatory agent in several pharmaceutical preparations in Germany, which subsequently were withdrawn from the market. The active principle of the plant extract A A are 0

a) in vivo b) xanthine oxidase c) zinc, pH 5.8

O

/~N,~'~NH

~

NA

o~ OH

4

d G - A A I (R=OCH3) dG-AAII (R=H)

R

N

HoH3 OH dA-AAI (R=OCH3) dA-AAII (R=H)

Fig. 1. Proposed mechanism of DNA adduct formation by AAI (1, R = OCH3) or AAII (1, R = H) reductively activated in vivo or in vitro. The four major AA adducts formed are dA-AAI (3, R = OCH3), dA-AAII (3, R = H), dG-AAI (4, R = OCH3) and dG-AAII (4, R = H).

T.H. Broschard et al. / Cancer Letters 98 (1995) 47-56

the aristolochic acids; aristolochic acid I (AAI) and aristolochic acid II (AAII) being the major constituents. Both acids are nitrophenanthrene derivatives differing from one another by one methoxy group at position 8 (Fig. 1). AAI and AAII as well as the natural mixture AA have been found to be mutagenic in various shortterm tests [3-6]. In the Ames test the mutagenic activity of AAI and AAII depends on the presence of bacterial nitroreductases in Salmonella typhimurium strains and base substitution mutations [7] are primarily induced. Metabolism studies [8,9] confirmed that AAI and AAII are activated by a reductive pathway via a cyclic N-acylnitrenium ion with delocalized positive charge (Fig. 1). The major DNA adducts of AAI and AAII formed in vitro and in vivo have been characterized, showing that both acids bind preferentially to the exocyclic amino group of purine nucleotides and contain the fluorescent aristolactam moiety [10]. The unique characteristic of these DNA adducts is that the purine bases are linked to the carbon atom ortho to the lactam nitrogen. Both deoxyadenosine adducts, 7(deoxyadenosin-N~-yl)-aristolactam I (dA-AAI) and 7-(deoxyadenosin-N~-yl)-aristolactam II (dA-AAII) exhibit imino character, while both deoxyguanosine adducts, 7-(deoxyguanosin-N2-yl)-aristolactam I (dGAAI) and 7-(deoxyguanosin-N2-yl)-aristolactam II (dG-AAII) exhibit a secondary amine type bonding. Utilizing the 32p-postlabeling assay we showed that dA-AAI exhibit an apparently lifelong persistence in forestomach, the target organ of AAIinduced carcinogenicity in rats, whereas dG-AAI was removed continuously over a 36-week period [11]. Earlier studies on oncogene activation in AAIinduced rat tumours revealed a high incidence of AT --->TA transversion mutations in codon 61 of the H-ras gene. This apparent selectivity tor mutations at adenine residues is consistent with the extensive formation of dA-AAI adducts in forestomach DNA of rats 112]. In this context, oligonucleotides containing defined and site-specifically placed adducts are useful tools in explaining how individual chemical lesions formed in DNA by carcinogens are converted into mutations found in target genes of carcinogenesis, e.g. cellular oncogenes [13-15]. Recently we described the synthesis of 18-mer oligonucleotides

49

containing the major DNA adducts formed by the plant carcinogen AA in vivo located at a defined site. These adducted oligonucleotides were used as templates in primed DNA replication reactions with T7 DNA polymerase (Sequenase) and provided a basis for understanding the activation of ras protooncogenes by AT --->TA transversion mutations in rodent tumours by AA [16]. Although DNA synthesis by Sequenase was blocked predominantly at the nucleotide 3' to each adduct, the deoxyadenosine adducts allowed a small amount of translesional synthesis as well as incorporation of dAMP equally well as dTMP opposite the adducts. These studies have now been extended by using a eukaryotic polymerase human pol a. To this end adducted 30-mer oligonucleotides were synthesized, thereby making possible the use of longer primers (23-mers) in primer extension reactions at 37~ the optimal temperature for mammalian DNA polymerases [17]. We show that dA-AAI, dGAAI and dA-AAII are strong blocks of replication. Only dG-AAII allows elongation past the adduct site until the end of the template presumably by incorporation of the correct nucleotide (dCMP) opposite the adduct. 2. Materials and methods

2.1. Materials Pure AAI and AAII as sodium salts were kindly provided by Fa. Madaus (Koeln, Germany). [y 32p]_ ATP (specific activity >7000 Ci/mmol) was obtained from ICN Biomedicals. Human DNA polymerase a was purchased from Molecular Biology Resources, Milwaukee, WI, and T4 polynucleotide was from AGS GmbH (Heidelberg, Germany). Ultrapure grade deoxyribonucleoside-5'-triphosphates and Sephadex G-25 were purchased from Pharmacia P-L Biochemicals. Enzymes and chemicals for the 32p-postlabelling assay were obtained commercially from sources described before [16]. All other chemicals were of high grade quality and were purchased from different sources.

A Bischoff HPLC pump equipped with a fluorescence-detector (Bischoff, Monitor 8000) and a UVdetector (Latek, spectroMonitor III) was used for separation and analysis of modified and unmodified oligodeoxynucleotides.

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T.H. Broschard et al. / Cancer Letters 98 (1995) 47-56

Table 1 Sequences of oligodeoxynucleotides OligoA30 OligoG30 Prim23 Prim26

5'I-I-rATTTTTTCCCCTTCTTTCCTCTTCCCT3' 5'TTTGqTITVrCCCCTTCTI~CCTCTTCCCT3' 5'AGGGAAGAGGAAAGAAGGGGAAA3' 5'AGGGAAGAGGAAAGAAGGGGAAAAAA3'

Different oligonucleotides (see Table 1) were chemically synthesized (Applied Biosystems, model 380 B) and purified by reversed phase HPLC.

2.2. Preparation of modified oligodeoxynucleotides Oligodeoxynucleotides containing single dA-AAI, dG-AAI, dA-AAII or dG-AAII lesions were prepared, isolated and characterized as described previously [16]. Briefly, a 100/zM solution of either oligoA30 or oligoG30 (concentration of single strands) in 50 mM potassium phosphate buffer (pH 5.8) was incubated with either AAI or AAII as sodium salts (molar ratio [carcinogen]/[DNA] = 20:1) for 6 h at 37~ in the dark, using zinc as an activating system. The modified oligodeoxynucleotides were separated from unmodified DNA strands by reversed phase HPLC (linear gradient from 5 to 50% (v/v) acetonitrile in 0.1 M TEAA, pH 7.0, over a 90 min period). Fractions containing adducted 30-mers, which were easily detectable by their fluorescent properties (Ex 360 nm, Em 490 nm), were collected, dried in a Speed Vac Concentrator and repurified by HPLC using different isocratic elution conditions (acetonitrile in 0.1 M TEAA (pH 7.0): 17% for oligoA30AAI; 16% for oligoA30-AAII; 15% for oligoG30AAI and oligoG30-AAII). The purity and identity of the four AA-monoadducted oligodeoxynucleotides was established by denaturing PAGE (20%, 7 M urea) and 32p-postlabelling, as described previously in detail [16].

2.3. Primer extension reactions Oligodeoxynucleotide primers prim23 and prim26 were end-labelled with [y-32p]ATP and T4-PNK and purified on a Sephadex G-25 column. Primed templates were obtained by annealing either the sitespecifically adducted or control 30-mer templates (0.13/~M) to the complementary 5'-32p-labelled 23-

mer or 26-mer primers (0.13/zM) in 105 mM TrisHCI (pH 8.0) and 8.8 mM Mg-acetate. This mixture was heated to 72~ for 5 min and then slowly cooled down to 4~ for annealing to occur. This annealed template-primer mixture (6.27 vols.) was mixed with 1 vol. of a solution containing 1.4 mM DTT, 0.14 mM spermine tetrachloride and 0.4mg/ml bovine serum albumin (BSA). Then 0.73 vol. of human polymerase a (1.5 units//zl) was added to make the polymerase-template-primer mixture. Polymerase reactions were initiated by adding 8/zl of the mixture containing enzyme-primer-template complex to 3/zl of dNTP dissolved in water. With prim23, combinations of all four dNTPs were used, whereas experiments with prim26 were conducted with individual dNTPs. Final concentrations of dNTP were either 50, 200, 400 or 600/~M. Other final concentrations for pol a-reactions: 5 mM Mg-acetate, 60mM Tris-HC1 (pH 8.0), 1 mM DTT, 0.1 mM spermine tetrachloride, 0.3 mg/ml BSA, 0.1 u//~l pol a, 0.07/zM template and 0.07/zM primer. Polymerase reactions were kept at 37~ for 75 min and were terminated by the addition of 6/zl of a solution containing 95% formamide, 20mM EDTA, 0.05% bromophenyl blue and 0.05% xylene cyanol and cooling. After heating at 90~ for 2 min, 2/zl aliquots were loaded on 20% polyacrylamide denaturing sequencing gels. After electrophoresis the gel was dried and radioactive bands were quantified by Phosphorlmaging (Molecular Dynamics). 3. Results

3.1. Preparation of site-specifically adducted oligodeoxynucleotides The synthesis of adducted oligodeoxynucleotides (18-mers) by AAI and AAII activated by chemical reduction with zinc have been reported [16]. Here we reacted 30-mers containing only a single purine base (Table 1) with AAI or AAII and zinc and analysed the reaction mixtures by HPLC using a fluorescence detector. As an example, the analytical HPLC elution profile of the AAI/oligoA30 reaction mixture is shown in Fig. 2: the fluorescence detected peak eluting at 30.9 min (Fig. 2D) represents the AAImodified oligoA30 (oligoA30-AAI), which is almost

T.H. Broschard et al. / C a n c e r Letters 98 (1995) 47-56

undetectable under UV absorption (Fig. 2B). Besides some as yet unidentified material, the reductive metabolite of AAI, the highly fluorescent lactam (AlacI), eluted at 73.4 min. The same HPLC conditions were used to separate the other adducted 30mers: oligoA30-AAII, retention time 27.5 min; oligoG30-AAI, retention time 25.5 rain and oligoG30AAII, retention time 23.9 min. As expected oligonucleotides bearing a guanine adduct eluted faster than oligonucleotides with an adenine adduct and AAIIadducted oligonucleotides eluted faster than AAIadducted ones. Fractions containing these modified oligomers were collected, pooled and subjected to a second HPLC purification. By comparison of peak areas relative to the unmodified 30-mers the yield of adducted oligomers was estimated to be approximately 0.5-1%.

A

E 0 ~0 CY o c 0

;BoligoA30

\

..Q

>

~

f

3.2. Characterization of adducted oligodeoxynucleotides

C Alacl E

c 0 O~ ~r

E c 0

m

51

D

$

The purities of site-specifically adducted oligonucleotides were determined by denaturing gel electrophoresis and 32p-postlabeling analysis as reported [ 16]. It is noteworthy that the adducted four 30-mers did not contain any visible contamination of the unmodified 30-mers (data not shown). 32P-Postlabeling analysis of the four adducted oligomers gave rise to a single bisphosphate adduct spot, which was identified by co-chromatography with authentic markers of the four known DNA adducts dA-AAI, dG-AAI, dA-AAII and dG-AAII [18].

oligoA30- AAI

3.3. Replication by human polymerase a 0

min

i

i

i

20

40

60

Fig. 2. HPLC elution profiles of the reaction mixture of oligoA30 with AAI/Zn in phosphate buffer, (pH 5.8) (B,D) and the control incubation consisting of AAI/Zn in phosphate buffer (pH 5.8) (A,C). Detection was by UV absorption (A,B) and fluorescence (C,D). HPLC conditions: linear gradient of 5-50% acetonitrile in 0.1 M TEAA buffer (pH 7.0) over a 90 min period, flow rate 1 ml/min. Retention times: oligoA30, 20.7 min; oligoA30-AAI, 30.9 min; aristolactam I (Alac 1), 73.4 min. The position of oligoA30-AAI is marked by the dotted line.

DNA polymerase a (pol a) is a major DNA replication enzyme of mammalian cells [19]. Its primary role is to provide an initiator DNA for subsequent DNA synthesis by DNA polymerase fl for the lagging strand [20]. Primer extension studies were undertaken with pol a to evaluate adduct-specific replication by a eukaryotic enzyme in comparison to results obtained by a bacteriophage DNA polymerase (Sequenase) reported earlier [ 16]. Both the AA modified 30-mers and the unmodified 30-mers (oligoA30 and oligoG30) as controls were hybridized to a 5'-32p-labelled complementary 23-mer primer prim 23 (Fig. 3). Each of the different

52

T.H. Broschard et al. / Cancer Letters 98 (1995) 47-56 dNTl's

AGGGAAGAGGAAAGAAGGGGAAA Ir TCCCTTCTCCTTTCTTCCCCTTTTTT•

oligoA30-

[ -AAI

-AAII

1 112341

1234

C

oligoG30C

I

-AAI

-AAII

1 [ 123411234

I T

prim23

prim23

i

Fig. 3. Autoradiogram of PAGE-separated DNA products generated by extension of the 32p-labelled primer prim23 as shown schematically above. The 30-mers used as templates carried dAdo, dGuo, dA-AAI, dA-AAII, dG-AAI or dG-AAII at position 27 from the 3' end (marked as X in the scheme). Primed templates were incubated with human polymerase a for 75 min at 37~ in the presence of M2+ (5 mM) and all four dNTPs at 50#M (1); 200/~M (2); 400/~M (3); and 600/zM (4). C, oligoA30 or oligoG30; prim 23, primer. Other conditions have been described under experimental procedures.

was as strong as by the A A I adducts and fully extended primers were almost undetectable the site of pol a arrest was clearly dependent on the dNTP concentration. As shown in Fig. 3, higher dNTP concentrations resulted in increasing amounts of 27 and 28 nucleotide products indicating blocking opposite and one base 5' to the dA-AAII adduct. In contrast, pol a displayed a totally different replication profile when dG-AAII adducted templates were used. Termination of primer extension 3' to this adduct was still the predominant reaction but translesional synthesis to fully extended primers was substantial and increased from 18% at 5 0 # M dNTP to 24% at 600/~M dNTP (Fig. 4). The results demonstrate that the purine adducts of A A I and AAII, present in a synthetic DNA template, predominantly block DNA replication by polymerase a, but that the site of termination is depending on the adduct strucpol ot (human)

i i i 5o~M AA-adducts is present at template position 27 from the 3' end. Since the previous studies with Sequenase were conducted at 25~ we compared the action of pol a at 25 and 37~ in primer extension reactions on the two unmodified 30-mers (oligoA30 and oligoG30). Using 50/zM dNTP, reactions were stopped either at 5 or 45 min and analysed by denaturing gel electrophoresis (data not shown). The results demonstrated that with both templates complete primer extension (to 30-mer products) occurred only at 37~ and. after 45 min. The extension product profile obtained at 25~ after 45 min was similar to the profile at 370C after 5 min. Consequently, all primer extension reactions with pol a described here were performed at 37~ for 75 min. As illustrated in Fig. 3, primer extension stopped almost completely one base 3' to the damage when the damage represented an A A I adduct (dA-AAI or dG-AAI). Increasing dNTP concentrations from 50 to 600/zM had only a small effect on bypass synthesis on these two adducted templates and even with 600 # M dNTP translesional synthesis remained negligible (Fig. 4). Both A A I lesions blocked primer extension to a small extent <5% also opposite the adduct creating 27-mer base products. Although blocking of DNA synthesis by dA-AAII

100%

i~

600 p.M

[

80% 60% 40%

JJ

20%

E

E

E

E

E

E

oligoA30-AAI

~

E

E

E

oligoA30-AAIl

100% 80% 60%

mlm

40% 20% 0%

E

oligoG30-AAI

E

E

E

E

oligoG30-AAII

Fig. 4. Quantitative analysis of DNA synthesis products formed by extension of the 23-mer primer and different templates as shown in Fig. 3. For clarity only products at 50/zM dNTP (dark coloured columns) or 600/tM dNTP (light coloured columns) are shown, as indicated. Each column represents the mean of two experiments.

53

T.H. Broschard et al. / Cancer Letters 98 (1995) 47-56

dNTP

AGGGA A G A G G A A A G A A G G G G A A A AA A ,#// T C C C T T C T C C T T T C T T C C C C T T TT T T X T T T

oligoA30

I control I-AAI

oligoG30

I -AAII I

P IA r GC I IA T G C I IA TGC I

I control I-AAI

I ATGc I IATGCl

I -AAII I

IA TGC I

Fig. 5. Autoradiogramof PAGE-separatedDNA products formedby extension of a 26-merprimer by polymerasect and a 400/tM concentration of only one dNTP for 75 min at 37~ The 30-mer templates contained either the normal purine bases or adducted purine bases at position X as indicated. The dNTPs used are shown as A, T, G, and C for dATP, dTTP, dGTP and dCTP, respectively. Lane P, 26-mer primer. ture. Moreover, only the dG-AAII adduct was bypassed by polymerase ct with high efficiency. 3.4. Identification of bases incorporated opposite dAAAL dG-AAI, dA-AAH or dG-AAH by pol a

Since substantial translesional synthesis occurred with dG-AAII and dA-AAII adducted templates and small amounts of incorporation of a nucleotide opposite the AA lesions (27-mers) was found in all cases, it was of interest to determine which nucleotide was preferentially placed opposite each of the four AA lesions. To this end, a 26-mer primer was used so that the first nucleotide to be incorporated by pol a would be immediately opposite the adduct at the 27th base from the 3" end of the templates. The primer-template complex was incubated with the polymerase, Mg 2§ and just one nucleotide triphosphate at 400 HM for 75 min. Using the unmodified oligoA30, pol a incorporated preferentially dTMP across adenosine as expected but also dATP and to a lesser extent dCTP led to the extension of prim26 (Fig. 5). A small proportion of incorporation of

dTMP occurred twice due to the fact that the first incorporation of dTMP represents the normal Watson-Crick base pair. In contrast no primer extension on the dA-AAI adducted template was detectable regardless of the nucleotide triphosphate used. This indicates a complete inhibition of pol a under these circumstances, which was not observed when all 4 dNTPs were present. It is clear that dA-AAII allowed the incorporation of dTMP and small amounts of dAMP by pol a. Using oligoG30 as template, pol a was able to extend each of the nucleotide triphosphates. While extension by dCTP was almost quantitative, only negligible extension by dTI'P and dGTP was observed. OligoG30-AAI as template blocked primer extension to a large extent. However, it was clear that dCMP and dAMP were incorporated opposite the dG-AAI adduct. Quantitative analysis revealed that the ratio of incorporation dCMP/dAMP was 10:1 in favour of the correct base pair. Likewise preferential incorporation of the correct nucleotide dCMP was observed with the dG-AAII adduct in template oligoG30-AAII.

54

T.H. Broschard et al. /Cancer Letters 98 (1995) 47-56

4. Discussion

In order to understand how the plant carcinogen AA and related nitroaromatics can give rise to mutations, an in vitro investigation was undertaken to study the action of a eukaryotic DNA polymerase (human pol a) upon encountering these bulky AA adducts and compare the results with those obtained previously by a prokaryotic polymerase (Sequenase). Since the templates used in both studies consist of the same sequence context and adduct site, they are well suited for comparing the properties of the two polymerases. As expected, human pol a fully extended primers on unadducted 30-mer templates only when incubations were conducted at 37~ Consequently, the shorter primer template complexes (ll-mer primer/ 18-mer template) used in our experiments with Sequenase were not suitable due to their melting temperature of 30~ [21]. Therefore site-specifically adducted 30-mer oligomers were prepared by in vitro reactions following the approach outlined by Reardon et al. [22] and described before [16]. The purity and identity of the four AA adducted 30-mers under study were determined by HPLC, gel electrophoresis and 32p-postlabeling analysis. The polymerase arrest site confirmed the site of adduction of the oligonucleotides. To characterize the effects of bulky adducts on DNA replication, several in vitro studies have been performed and have shown that the effects are a function of the size, site, and stereochemistry of the adduct, the sequence context of the DNA template, and the nature of the polymerase used [15,23-35]. Moreover, these studies demonstrated that DNA adducts have four fates during replication: (i) blockage of DNA synthesis one base 3' to the adduct, and/or (ii) opposite the adduct, (iii) translesional synthesis to either full length of the template, or (iv) termination of synthesis after translesion replication but prior to complete primer extension. In agreement with other studies as well as our recently reported results with Sequenase, most of the DNA synthesis on AA adducted templates was terminated immediately 3' to the adducts. As illustrated in Fig. 4 at 5 0 ~ M dNTP this effect was strongest on templates containing dA-AAI and decreased in the order of dA-AAI > dG-AAI > dA-AAII >> dG-AAII.

Examination of computer generated models reported previously [16] revealed that strong stacking with the adenine residue at the 3' end of the primer prim26 can explain this blocking activity by AA adducts. Incorporation of dNMP opposite the lesion is inhibited because this space is occupied by the aristolactam ring regardless of the different structures of AA adducts. Although the replication profile obtained by the two AAI adducted templates is quite similar there is clear evidence that dAdo adducts are more effective blocks to replication than dGuo adducts indicated by the smaller amount of fully extended primers. This observation is also in agreement with our previous study and might be due to the stronger rigidity of the imino structure in the N6-dAdo adducts compared to the amino structure in the N2-dGuo adducts (Fig. 1). The effect of an additional methoxy group at the aristolactam moiety in the AAI adducts, which sticks out of the aromatic plane was more pronounced in the pol a catalysed reactions than in the reactions catalysed by Sequenase. Since sequencing analysis on the 30-mers obtained especially with the oligoG30-AAII has not been carried out, the mechanisms by which pol a synthesized past the specific adducts is not known. Thus a bypass mechanism mediated by mispairing adjacent to the adducts which can result in non-targeted mutations, cannot be excluded [34]. Some small amounts of translesional synthesis products shorter than 30-mers were also detected in incubations of all four adducted templates. These products may be interpreted as the result of the formation of base deletions, as shown for the bulky benzo[a]pyrene-DNA adducts by Shibutani et al. [28] or premature dissociation of the polymerase reported by Vyas and Basu [37]. The differences in polymerase action between pol a and Sequenase on AA adducted templates is further exemplified by nucleotide incorporation studies. With pol a no introduction of any nucleotide opposite dA-AAI was observed. On the other hand the same lesion allowed the incorporation of dTMP, resuiting in a non-mutagenic event, to the same degree as the incorporation of dAMP, resulting in A--~ T transversion mutations by Sequenase [16]. The other three AA lesions led to the preferential incorporation of the correct base and to a lesser extent dAMP when

T.H. Broschard et al. / Cancer Letters 98 (1995) 47-56

pol a was the polymerase. This is consistent with results reported by Chary and Lloyd [35] for polymerases that lacked 3' ~ 5' exonucleolytic activity. The t e r m i n a t i o n of D N A synthesis at a defined distance after translesion replication but before reaching the end of the template has first been reported by R e a r d o n et al. [22] with Sequenase and later then with HIV-1 reverse transcriptase [36], pol a [33] and K l e n o w f r a g m e n t of D N A polymerase I [37] with various b u l k y lesions. Replication by pol a at 6 0 0 / ~ M d N T P on d A - A A I I templates also revealed substantial t e r m i n a t i o n one base 5' to this lesion (Fig. 4), indicating that this specific adduct can block primer extension even w h e n positioned in the template-primer stem. In contrast, Sequenase did not give rise to such p r i m e r extension products under similar i n c u b a t i o n conditions. W e do not k n o w the reason for this difference. However, there are some differences b e t w e e n these two polymerases, of which perhaps the most important is that pol a has a m u c h lower processivity. T a k e n together, these data suggest that A A - a d d u c t s represent either strong blocks of D N A replication for pol a or w h e n translesion bypass by pol a occurs it is primarily error free.

Acknowledgements W e thank Dr. R.P.P. Fuchs for a critical review of the manuscript. This work was supported by a stip e n d for H.H.S. from the Deutsche F o r s c h u n g s g e meinschaft.

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