Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector DNA transfected into monkey cells

24

Fundamental and Molecular

Mechanisms of Mutagenesis

ELSEVIER

Mutation Research 308 (1994) 117-125

Mutation spectrum of 4-nitroquinoline 1-oxide-damaged single-stranded shuttle vector D N A transfected into monkey cells Gilberto Fronza a,,, Catherine Madzak b, Paola Campomenosi a, Alberto Inga a, Raffaella Iannone a, Angelo Abbondandolo a,c, Alain Sarasin b a CSTA-Laboratory of Mutagenesis, National Institute for Research on Cancer (IST), Hale Benedetto XE, 10, 1-16132 Genoa, Italy, b Laboratory of Molecular Genetics, Institut de Recherches Scientifiques sur le Cancer, P.O. Box 8, F-94801 Villejuif cedex, France, c Chair of Genetics, University of Genoa, Italy Received 22 June 1993; revision received 17 December 1993; accepted 21 December 1993

Abstract

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen which induces two main guanine adducts at positions C8 and N2. We recently determined the mutation spectrum induced by the ultimate metabolite of 4NQO, acetoxy-4-aminoquinoline 1-oxide in the M131acZ'/E. coli lacZzlM15 a-complementation assay. Our data suggested that dGuo-C8-AQO induces (per se or via AP sites) G to Pyr transversions. Here we report our study on 4NQO mutagenesis in monkey cells. 4NQO lesions were induced in vitro on a single-stranded (ss) DNA shuttle vector carrying the supF tRNA gene. This vector was able to replicate both in mammalian cells and in bacteria. The mutations induced in monkey cells were screened by the white/blue /3-galactosidase activity assay in E. coli. We took advantage of the peculiar feature of ss supF DNA in which the extent of secondary structure may be a function of the temperature, with the dependence of the 4NQO-specific adduct spectrum on DNA secondary structure. We reasoned that mutational spectra derived from damage induced in the presence (20°C) or absence (70°C) of DNA secondary structure should be different. The result of sequencing a total of 89 induced and spontaneous mutants confirmed that the spectra are statistically different. These data suggest that the two 4NQO guanine adducts may induce different mutations.

Key words: 4-Nitroquinoline 1-oxide mutation spectrum; Shuttle vector; SupF 1. Introduction * Corresponding author. Tel. 39.10.3534292; Fax 39.10.352999. Abbreviations: 4NQO, 4-nitroquinoline I-oxide; Ac-4HAQO, acetoxy-4-aminoquinoline 1-oxide; dGuo-N2-AQO, 3-(deoxyguanosin-N2-yl)-4-aminoquinoline 1-oxide; dGuo-C8AQO, N-(deoxyguanosin-C8-yl)-4-aminoquinoline 1-oxide; dAdo-N6-AQO, 3-(deoxyadenosin-N6-yl)-aminoquinoline 1oxide; AP, apurinic/apyrimidinic; Me2SO, dimethyl sulfoxide; X-Gal, 5-bromo-4-chloro-3-indolyl-/3-o-galactowranoside; IPTG, isopropyl-/3-D-thiogalactoside; ss or ds DNA, single- or double-stranded DNA; pyr, pyrimidine; Amp, ampicillin.

4-Nitroquinoline 1-oxide (4NQO) is a potent mutagen and carcinogen inducing tumors particularly in lung, pancreas and stomach (Sugimura, 1981). It shows the peculiar feature of having both UV-mimetic (in prokaryotes and eukaryotes) and X-ray-mimetic (only in eukaryotes) properties (Bailleul et al., 1989). Although extensively used in carcinogenesis studies, its mechanism of mutation induction is far from being elucidated at

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G. Fronza et al. / Mutation Research 308 (1994) 117-125

the molecular level. 4NQO, after metabolic activation, binds to DNA producing three main adducts: two on guanine (dGuo-N2-AQO, dGuoC8-AQO) and one on adenine (dAdo-N6-AQO) (Bailleul et al., 1981; Gali~gue-Zouitina et al., 1986). The relative proportion of these adducts is identical in in vivo experiments in rats using 4NQO, and in in vitro experiments using ds DNA damaged with the ultimate metabolite of 4NQO, Ac-4HAQO (Gali~gue-Zouitina et al., 1985). In both cases dGuo-N2-AQO, dGuo-C8-AQO and dAdo-N6-AQO represent about 50%, 25% and 10% of total 4NQO adducts, respectively. If ss DNA is damaged in vitro with Ac-4HAQO, dGuo-C8-AQO represents quantitatively the major adduct (75-80%), while the other two adducts represent only minor lesions (Gali~gue-Zouitina et al., 1984). Evidence suggesting that dGuo-N2AQO and dGuo-C8-AQO may cause different structural changes at the lesion site has been reported (Bailleul et al., 1984; Fronza et al., 1990; Panigrahi and Walker, 1990). These differences may suggest that the two adducts have different mutational specificity. We recently reported that the 4NQO mutational spectrum obtained from a damaged ss phage DNA transfected into E. coli was characterized by a high proportion of guanine to pyrimidine transversions, indicating a possible association between dGuo-C8-AQO and this type of mutations (Fronza et al., 1992). Recently, SV40-based shuttle vectors that can be used as ss DNA in eukaryotic cells have been described (Madzak et al., 1989; Madzak and Sarasin, 1991a,b; Cabral-Neto et al., 1992). In a system where both ss and ds DNA vectors were transfected into monkey cells, the type and location of UV-induced mutations were found to be very similar although some different classes of mutations could be observed (Madzak and Sarasin, 1991a; Madzak et al., 1992). In the present study, we intended to verify if the ss DNA-specific mutational spectrum observed with 4NQO in E. coli (Fronza et al., 1992) could also be reproduced in an eukaryotic system. For this purpose, we used the pZ189 shuttle vector (Seidman et al., 1985; Kraemer and Seidman, 1989) and African green monkey kidney cells (CV1P). The plasmid pZ189 contains the

origin of replication and the large T-antigen gene of SV40 and the supF target gene, which encodes a suppressor tRNA. In addition, this plasmid contains the intergenic region from M13 phage coding for the sequences required for the production of ss plasmid DNA in M13-infected cells (Zagursky and Berman, 1984). Interestingly, in the ss supF DNA, the extent of ~econdary structure may be a function of the temperature. Since the 4NQO adduct spectrum depends on DNA conformation (Gali~gue-Zouitina et al., 1984), we wanted to determine the mutational spectra derived from damage induced in the presence (20°C) or absence (70°C) of secondary structure. Results will be discussed in the light of the hypothesis that the two 4NQO guanine adducts may induce different mutations. 2. Materials and methods

Cells and bacterial strains African green monkey kidney cells (CV1P) (Sarasin and Benoit, 1980) were grown in Dulbecco's modified Eagle's medium with 7% fetal calf serum and antibiotics at 37°C in a 5% CO2 atmosphere. The bacterial E. coil strain MBM 7070, laCZam CA7020 lacY1, hsdR-, hsdM ÷ araD139 AaraABC-leu7679 galU galK rpsL thi, was used as host for pZ189, a 5479-bp DNA plasmid (Seidman et al., 1985). The E. coli host strain is Amp s and carries an amber mutation in its lacZ gene. Thus bacteria harboring pZ189 plasmid can be selected for resistance to ampicillin and the functionality of the amber suppressor tRNA gene can be easily detected by testing /3-galactosidase activity on X - G a l / I P T G / A m p plates. Plasmid pZ189 ds DNA was prepared using a standard procedure (Sambrook et al., 1989). pZ189 ss DNA was prepared in E. coli FG1 strain (r- m ÷ Alac pro F'lac) as previously described (Madzak et al., 1989) using M13K07 or R408 (Pharmacia-PL, Biochemicals Inc.) as helper phages. Purification of the ss pZ189 was carried out on preparative low-melting agarose gel. The band corresponding to ss pZ189 DNA was excised and DNA was electro-eluted, precipitated with ethanol and checked for purity by electrophoresis.

G. Fronza et al. / Mutation Research 308 (1994) 117-125

Chemicals and enzymes IPTG and X-Gal were obtained from Boehringer Mannheim, Germany. Sequencing kit (Sequenase version 2.0) was obtained from USB, USA. 4NQO was obtained from Sigma, [a35S]dATP, specific activity > 1000 Ci/mmole, from Amersham. Ac-4HAQO was synthesized as previously described and its purity checked by HPLC (Bailleul et al., 1983). DNA modification DNA samples (ss or ds pZ189) were modified in vitro by Ac-4HAQO in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 4% Me2SO at the indicated concentrations and purified by three ethanol precipitations as previously described (Menichini et al., 1989). Since the supF gene may exhibit potential secondary structures as suggested by Kraemer and Seidman (1989) and by analysis of its sequence with a computer program predicting secondary structures in ss DNA (PC-software: Oligo, version 3.4, by Wojciech Richlik), we damaged ss pZ189 at 20°C for 15 min (where DNA secondary structure should exist) or at 70°C for 2 min. In the latter situation the gene target should be completely single stranded. The number of adducts per ss pZ189 molecule was extrapolated from standard curves obtained using the radiola-

119

belled compound as previously described (Fronza et al., 1992).

DNA transfection and plasmid recovery Undamaged or Ac-4HAQO-damaged pZ189 DNA (ss or ds) was transfected into CV1P cells using the DEAE-Dextran technique (Wilson, 1978). After 2 days, extrachromosomal DNA was recovered from transfected cells by a small-scale alkaline lysis procedure (Stary and Sarasin, 1992). Shuttling into bacteria and mutagenesis analysis Before shuttling into bacteria, rescued plasmids were digested with DpnI (20 units) to inactivate any original input plasmid (Peden et al., 1980). Plasmid DNA was shuttled into MBM7070 E. coli cells by electroporation using a CellJect apparatus (Eurogentec). Transformants were plated on LB Petri dishes supplemented with 12.5 ~ g / m l ampicillin, 0.2 mg/ml X-Gal and 20 mg/l IPTG. Cells containing plasmids with a functional supF gene, which suppresses the lacZam mutation, can form blue colonies on A m p / X Gal/IPTG plates whereas cells containing plasmids with mutated, inactive supF gene form white or light blue colonies. Each mutant colony was picked, replated on A m p / X - G a l / I P T G plates and, after phenotype confirmation, 4-ml samples

Table 1 Survival and mutation induction of undamaged or Ac-4HAQO-damaged ss or ds pZ189 in MBM7070 or after passage through CV1P cells DNA

sspZ189

dspZ189

Treatment

supF mutant frequency a

Ac-4HAQO

Survival (%)

(p.M)

MBM7070

CV1P

CV1P 144/38740 (3.7 × 10 -3) b 218/5884 (37 × 10 -3) 127/4588 (28 × 10 -3)

20°C, 15 rain

0

100

100

20°C, 15 rain

9

70°C, 2 min

9

0.72 + 0.45 (n = 5) 0.73 + 0.12 (n = 5)

23.6 + 8.7 (n = 5) 26.4 + 1 0 . 7 (n = 5)

20°C, 15 min

0

100

100

20*(2, 15 min

90

7.4 + 1.6 (n = 5)

13.2 + 3.7 (n = 4)

18/16482 (1.09 × 10 -3) 21/1998 (10.5 × 10 -3)

a Inactivation of the supF gene was assayed as described in Materials and methods, given here as number of white or light blue colonies to total. b Spontaneous mutation frequencies at 20oC (15 min) and 70°C (2 min) were practically identical.

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G. Fronza et aL / Mutation Research 308 (1994) 117-125

of liquid culture (LB with Amp) were grown overnight. Before plasmid extraction, each culture was streaked again on A m p / X - G a l / I P T G plates to assess phenotypic homogeneity. Mutant plasmids were purified with Quiagen columns according to the manufacturer's instructions (Diagen, Germany). Plasmids were first analyzed by agarose gel electrophoresis for the presence of large deletions or rearrangements. Plasmids were then sequenced by the chain termination method (Sanger et al., 1977) using as primer a 16 bases long oligonucleotide (5'-TAGGCGTATCACGAGG-3'), hybridizing to the terminal part of the Ampr gene, synthesized with an Applied Biosystem oligonucleotides synthesizer Model 380A.

survival of pZ189-damaged DNA is lower when transfected directly in MBM7070 than after passage through CV1P, especially for ss DNA. Similar results were obtained for UV-irradiated (200 J / m 2) ss pCF3A shuttle vector (Madzak et al., 1992). ss pZ189 damaged at 20°C or at 70°C showed identical survival, suggesting that the global adduction of the two damaging treatments was, as expected, very similar. However, ds DNA was more resistant than ss DNA to the toxic effects of Ac-4HAQO-induced lesions. This is probably due to the fact that excision repair processes can operate on ds DNA while they cannot operate on ss DNA. The decrease in survival of ss and ds DNA was associated with an increase of mutation frequencies at the supF locus (Table 1). The mutation frequency is defined as the ratio of white or light blue colonies to the total. No significant difference in spontaneous mutation frequencies was found between ss DNA mock-treated at 20°C (15 min) or 70°C (2 min) (data not shown), suggesting that heating (2 min at 70°C) was not mutagenic per se. A spontaneous mutation frequency significantly higher (p < 0.02, Fisher's exact test) for ss pZ189 (3.7 × 10 -3) than for ds pZ189 (1.09 × 10 -3 ) was observed, confirming previous results (Madzak and Sarasin, 1991a). The spontaneous mutation frequency found in this study for ds pZ189 is similar to that obtained by Bredberg and Nachmansson (1987). At the highest concentration of Ac-4HAQO (9 /,M) used with ss DNA, induced mutation frequencies were increased about 10-fold in ss pZ189 damaged at 20°C or 70°C. In ds pZ189, a higher concentration of

3. Results

Survival and induction of mutations To study 4NQO-induced mutations in CV1P cells, ss and ds pZ189 were treated in vitro with the ultimate metabolite of 4NQO, Ac-4HAQO. Damaged or undamaged vectors were transfected into monkey cells as described in Materials and methods. Two days later, replicated plasmids were harvested and used to transform E. coli MBM7070 to ampicillin resistance. The survival was defined as the ratio of bacterial colonies transformed with the DNA recovered from cells transfected with Ac-4HAQO-damaged vector to those obtained with untreated vector. Survival and mutation induction for damaged ss or ds pZ189 are shown in Table 1. It is evident that

Table 2 Analysis of mutants obtained in the progeny of pZ189 generated during replication in CVIP cells DNA

Treatment

sspZ189

20°C, 15 min 20°C, 1'5 min 70°C, 2 min

dspZ189

20°C, 15 min

Ac-4HAQO (tzM)

Mutated sites

supF mutants a analyzed

Plasmids with rearrangements b

Plasmids sequenced

Mutation characterization single

multiple

0 9 9

32 25 27

(2/32) (2/25) (0/27)

30 23 27

28 14 23

2 9 3

14 20 14

90

9

(0/9)

9

7

2

9

a Inactivation of the supF gene was assayed as described in Materials and methods. b Large alteration of plasmid size, analyzed by agarose gel electrophoresis ( < 150 bp).

G. Fronza et al. / Mutation Research 308 (1994) 117-125 Table 3 Analysis of sequence alterations generated in the supF gene by replicating A c - 4 H A Q O - t r e a t e d or untreated sspZ189 in CV1P cells Type of

Sponta-

Ac-4HAQO-induced

mutation

neous

ss pZ189

ss pZ189

(20°C, 15 rain)

(70°C, 2 min)

G~T G~C G~A C~T C~A C ---, G T~C T--)A T~G A~C -1C - 1T -1G rearrangements

2 1 12 5 8 1 1 1 1 2

9 9 5 4 2 3 1 1 2

15 7 6 -

Total mutations Total mutants

34 32

36 25

30 27

-

1 -

1 -

For ds pZ189, we sequenced nine induced mutants and found 11 mutations: seven GC to Pyr transversions and four GC to AT transitions.

121

tions were observed. Only 3 / 3 2 (9.4%) are Gtargeted while the majority (25/32; 78%) are C-targeted. By contrast, Ac-4HAQO-induced mutants, both with ss and ds pZ189, are mainly G-targeted (23/34, 68% for ss pZ189 damaged at 20°C; 22/30, 73% for ss pZ189 damaged at 70°C). These differences between spontaneous and induced mutations are statistically significant (p < 5 × 10 -5 at 20°C and p < 10 - 6 at 70°C, Fisher's exact test). For ds pZ189, 11/11 induced mutations were targeted at GC base pairs. Among induced mutations targeted at guanine residues, transversions exceeded transitions by a factor of 3.6 for ss vector treated at 20°C (all G-targeted mutations were transversions at 70°C) and 1.7 for ds vector. Interestingly, five transitions were found among 23 G-targeted mutations with ss

T T T A 40 ,r, 50 ~ 60 ,~ 70~ A * "~* ~J * '~ *~ C C TC~ACACCACCCCAAGC~CTCGCCGG'I~[~CCCTCGTCTGAGATTF 3' 1 Tb bAi~m bATe -Td T T C C C Th

'~

c

Ac-4HAQO (90 /zM) was needed to induce the same increase (Table 1). By picking up supF mutants at the highest concentrations used, about 90% of mutations are expected to be induced by the chemical treatment.

TG T TA G TT

80*

mids were isolated after transfection of ss or ds A c - 4 H A Q O - d a m a g e d or control vectors in C V I P cells. The fraction of mutant plasmids showing

large deletions/rearrangements was determined by agarose gel electrophoresis analysis. This fraction is marginal, globally representing less than 8%, while most of the mutations observed are point mutations. The majority of sequence alterations are due to single changes; multiple (two or more) mutations are observed more frequently in Ac-4HAQO-induced than in spontaneous mutants. A total of 89 plasmids (30 spontaneous and 59 induced) were sequenced (Table 3). Among 30 spontaneous ss-derived mutants, 32 point muta-

~L

90 *

~10J v. II *1/~

C

!

°

T T C Ae

T

fi 110 U *

120* v.I

AGA~AGTAC.aSTGAA~TI'CCA A ~ A G G A AGGGGGTGGTGG 5' Te Tc Aa ~an~'~T Qf '~ '~.C. TI El

C

Molecular analysis of mutants As shown in Table 2, 93 supF- mutant plas-

T

}/

,-8

T

T

T

-IO

Fig. 1. Location of the spontaneous and A c - 4 H A Q O - i n d u c e d point mutations on the supF gene of ss pZ189 vector transfected into monkey C V I P cells. The sequence of the supF gene shown (0 = transcription start site) is that of the strand present in ss pZ189 and therefore the A c - 4 H A Q O - d a m a g e d one. Spontaneous and induced mutations are indicated above and below the wild-type sequence, respectively. Mutations obtained after A c - 4 H A Q O treatment of the ss shuttle vector D N A for 2 min at 70°C are outlined. Multiple mutations (different mutations found in the same mut a nt ) are indicated with the same number (1, 2; for spontaneous) or the same letter ( a - n ; for Ac-4HAQO-induced). O t h e r mutations were located out of the reported 37-124 nucleotide sequence: the second mutation found in mut a nt 2 is a C ~ G at position 8; for mut a nt f, C ~ G, pos. - 15; for mut a nt i, C --~ A, pos. 8; at pos. 8 a single m u t a n t C ~ A was found in Ac-4HAQO-induced mutants. Sites with more than four mutations are real hot spots (n > 4; p < 0.02). Note that the reported sequence is written from 3' to 5'.

122

G. Fronza et al. / Mutation Research 308 (1994) 117-125

vectors damaged at 20°C (15 min) while none were found among 22 G-targeted mutations after treatment at 70°C (2 min); this difference is statistically significant (p < 0.03). These data could be consistent with the hypothesis that dGuo-C8AQO and dGuo-N2-AQO, formed in different proportions in DNA with different secondary structure, cause different mutations. The location of independent point mutations along the supF tRNA gene of pZ189 is reported in Fig. 1. The distribution of mutations reveals a non-random distribution among the available sites. From studies on ss shuttle vectors (Madzak and Sarasin, 1991a; Cabral-Neto et al., 1992; Madzak et al., 1992; this work) we know that in this region there are 18 Cs and 22 Gs at which base substitutions cause supF inactivation. We found 23 C-targeted spontaneous mutations (with a mean of 23/18 = 1.3 mutations/site), and 20 or 25 G-targeted Ac-4HAQO-induced mutation at 20 or 70°C, respectively (means 20/22 = 0.91 mutation/site at 20°C; 25/22 = 1.1 mutations/site at 70°C). It can be calculated that sites with at least four mutations/site are real hot spots ( p < 0.02). It has to be remembered that position 75, where we found nine induced point mutations, is within the anticodon region of the tRNA (Kraemer and Seidman, 1989).

4. Discussion In the present work we report the spectrum of mutations induced during passage of Ac4HAQO-damaged ss pZ189 shuttle vector DNA in CV1P ceils. It is known that N2 adducts are formed mainly in ds DNA while C8 adducts are produced mainly in ss DNA. The supF gene may have a secondary structure similar to that of its tRNA (Kraemer and Seidman, 1989) especially if it is single stranded. We analyzed the predicted presence of secondary structure, at least transiently formed, in ss pZ189 by computer analysis. It appeared that ss supF DNA should have a series of stems and loops which should be present at relatively low salt concentration ( < 100 mM) and at room temperature (ziG = - 2 3 . 8 kcal/ mole). We thus reasoned that, since ss pZ189 at

room temperature may exhibit some ds structure, the treatment with Ac-4HAQO at 20°C should favor the formation of dGuo-N2-AQO and some G to A transitions are expected. When the treatment is done at 70°C (predicted melting temperature: 56°C at 10 mM Na+), no ds secondary structure should be present and predominant formation of C8 adducts is expected. In this case, no G to A transition should appear in the mutational spectrum. By comparison of the two Ac4HAQO mutational spectra obtained after treatment of ss pZ189 at different temperatures, we found five G to A transitions over 23 G mutations for ss pZ189 damaged at 20°C, significantly different (p < 0.03, Fisher's exact test) from the zero G transitions over 22 G mutations obtained with ss pZ189 damaged at 70°C. In a small sample of mutants obtained from ds DNA, four G to A transitions out of 11 mutants were found. Although the statistical significance of these data is low due to the limited sample size, a trend consistent with the adduct-specific mutation hypothesis is evident. Moreover, sequencing data from 4NQO-induced H PRT mutants in CHO cells showed that 12 transitions were found among 34 G-targeted base substitutions (Inga et al., 1994). Nevertheless, the fact that some sites where G --, A transitions occurred seem located in a loop at 20°C may suggest that a more complex process than that indicated above could lead to this type of mutation. A possible influence of neighboring base sequence on mutagenesis may exist for dGuo-C8AQO in ss DNA, as recently proposed by Daubersies et al. (1992) for dGuo-N2AQO in ds DNA. The data obtained in monkey cells with the use of ss pZ189 shuttle vector are practically identical to those obtained in E. coli with the M131acZ'/lacZAM15 system (Fronza et al., 1992). The two approaches are different not only from the phylogenetic point of view, but also for the type of biochemical steps the DNA information has to go through before phenotypic selection takes place: in the supF approach, transcription is the only needed event, while in the intracistronic a-complementation approach both transcription and translation have to occur. This suggests that basic processing of Ac-4HAQO le-

G. Fronza et al. / Mutation Research 308 (1994) 117-125

sions on ss DNA is very similar in E. coli and simian cells. We wanted to confirm this finding by directly comparing the mutation spectrum obtained after passage of Ac-4HAQO-damaged ss pZ189 into monkey cells, with that obtained directly in E. coli MBM7070 strain. As found by others using UV-irradiated ds (Hauser et al., 1986) or ss shuttle vectors (Madzak et al., 1992), we were unable to isolate mutants by direct transformation of MBM7070 with Ac-4HAQOdamaged ss pZ189. This fact clears the field from the possibility that the mutations observed in this study are due to residual lesions remaining unrepaired during replication in CV1P cells and inducing mutations directly in E. coli. The use of ss shuttle vectors in studying the mechanisms of mutagenesis in mammalian cells seems particularly useful (Madzak and Sarasin, 1991a; Cabral-Neto et al., 1992; this work). Indeed, a s s DNA carrying bulky lesions cannot be processed by excision repair mechanisms and translesion synthesis is necessary to produce fully replicated ds progeny. The most interesting advantage of the ss shuttle vector approach is that it permits an unambiguous determination of the nature of the base in correspondence to which a mutation arises. For example, GC ~ AT transitions are the most common base substitutions observed in spontaneous mutations using ds DNA shuttle vectors (Hauser et al., 1987; Miller et al., 1984). Miller et al. (1984) suggested that these point mutations might result from both cytosine modification and guanine depurination. In this work, as in previous works of some of us (Cabral-Neto et al., 1992; Madzak et al., 1992), this ambiguity is overcome since we found that in ss shuttle vectors the major mutational events are targeted to cytosines, indicating that cytosine modification is a major cause of spontaneous mutations in ss DNA. This is in agreement with the notion that this spontaneous event is faster in ss than ds DNA (Lindahl and Nyberg, 1974). Interestingly, experiments in progress show that 30% of spontaneous mutations are due to uracil (deaminated product of C), since they disappeared after treating the ss DNA with uracil-Nglycosylase before transfection (Cabral Neto et al., in preparation).

123

Another feature of ss shuttle vectors is that point mutations constitute the majority of spontaneous mutations. For example, when undamaged ds pZ189 is passed in monkey cells, 30-40% of mutations involve large deletions a n d / o r gross rearrangements (Hauser et al., 1986), while with ss pZ189 this percentage drops to less than 8%. These data suggest that lesions leading to large rearrangements on ds DNA are probably lethal on ss DNA (e.g., single-strand breaks) or that ss DNA interferes with the cellular processes that lead to deletions or rearrangements. Studying multiple point mutations in a d s shuttle vector propagated in human cells, Seidman et al. (1987) suggested that they could be generated by a gap-filling, error-prone polymerase. In E. coli, multiple mutations were also observed in the UV-induced spectrum of the ss M13 phage DNA (LeClerc et al., 1984). They were attributed to an error-prone polymerase activity in SOS-induced bacteria. By contrast, Miller et al. (1984) proposed that each of the multiple mutations could be caused by a damaged base (cytosine deamination/guanine depurination). These authors proposed that base modifications would occur during the exposure of DNA to the acidic environment in a cytoplasm compartment during the transfection process. The multiple mutations observed by us after transfection of ss DNA can be due to an error-prone replication pathway acting on the ss template, resembling that postulated in SOS-induced E. coli (LeClerc et al., 1984). DNA replication on an Ac-4HAQO-treated ss template could possibly lead to the formation of gaps opposite lesions, if 4NQO lesions block DNA synthesis. Such a structure could be a substrate for an error-prone polymerase. It could also be a substrate for a putative post-replication repair process which would occur between two distinct plasmid molecules. We cannot, however, rule out the hypothesis that multiple mutations observed are the result of an excision repair process working on ds DNA after copying the ss template. In conclusion, we showed that the 4NQO mutation spectrum obtained after passage in CV1P cells of ss pZ189 was characterized by G to Pyr transversions and that G to A transitions appear under conditions where a partial or complete ds

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G. Fronza et aL / Mutation Research 308 (1994) 117-125

structure was predicted. This interpretation confirms the results previously obtained in E. coli. The hypothetical models proposed to explain how dGuo-C8-AQO may base pair with either an anti dAdo (explaining G to T transversion) or, in its syn conformation, with an anti dGuo (explaining G to C transversions) (Fronza et al., 1992) are in agreement with the present results. By comparing the Ac-4HAQO mutational spectrum obtained in ss pZ189 (this work) with the UV-induced mutational spectrum on the supF gene obtained in a similar experimental system (Madzak and Sarasin, 1991a), it is evident that each DNA-damaging agent shows its own specificity, which is basically identical to the one found in a prokaryotic ss-based mutational assay (Fronza et al., 1992; LeClerc et al., 1984). This suggests that the main events involved in the mutation fixation processes are evolutionarily conserved from bacteria to primate cells.

Acknowledgements We are grateful to Drs. A. Gentil, F. Bourre, A. Stary and P. Menichini for helpful discussion. P.C. and R.I. worked under a grant from the Italian Association for Research on Cancer (AIRC). C.M. held a fellowship from the "Ligue Nationale contre le Cancer" (Paris, France). This work was partially supported by the Italian Association for Research on Cancer and by the Commission of the European Communities (contracts: EV5V-CT91-0012 and EV5V-CT92-0227).

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