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DNA sequence analysis of mutations induced by N-2-acetylamino-7-iodofluorene in plasmid pBR322 in Escherichia coli
J. Mol. Biol. (1990) 213,239-246
D N A Sequence Analysis of Mutations Induced by N-2-Acetylamino-7-iodofluorene in Plasmid pBR322 in Escherichia coli George R. Hoffmann~f and Robert P. P. Fuchs Groupe de Cancdrogdn~se et de Mutagdn~se Moldculaire et Structurale Institut de Biologic Moldculaire et Cellulaire Centre National de la Recherche Scientifique 15 rue Rend Descartes, 67084 Strasbourg cedex, France (Received 11 September 1989; accepted 19 January 1990) The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but - 2 frameshifts and + 1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G ' C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-~-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.
nogen N-acetoxy-AAF (N-AcO-AAF). Just as DNA can be treated in vitro with N-AcO-AAF to form AAF adducts, treatment of DNA in vitro with N-hydroxy-N-2-aminofluorene (N-OH-AF) specifically forms AF adducts (Tang & Lieberman, 1983). The principal reaction site in DNA treated with N-AcO-AAF or N-OH-AF is the C-8 position of guanine (Evans et al., 1980; Kriek, 1965; Kriek et al., 1967; Westra & Visser, 1979). Despite the similarity in sites of reaction, AAF and AF differ from one another with respect to conformational changes that their adducts cause in DNA (Daune et al., 1981) and the spectrum of mutations that they induce (Bichara & Fuchs, 1985; Koffel-Schwartz et al., 1984). Diverse lines of evidence, including formaldehyde unwinding, pattern of hydrolysis by endonuelease $1, linear dichroism, circular dichroism, reaction with specific antibodies, and thermal stability, support an insertion-denaturation model, also called a base-
1. I n t r o d u c t i o n
The aromatic amines N-2-acetylaminofluorene (AAF$) and N-2-aminofluorene (AF) have been the subject of intensive investigation with respect to chemical and physical interactions with DNA and mutagenic mechanisms (Bichara & Fuchs, 1985; Daune et al., 1981; Koffel-Schwartz et al., 1984). AAF is a hepatic carcinogen whose carcinogenicity requires the metabolic activation of the parent compound to yield electrophilic metabolites. In order to circumvent the need for metabolic activation, many studies of AAF in non-mammalian systems are conducted with the direct-acting carcit Author to whom all correspondence should be addressed, at Department of Biology, College of the Holy Cross, Worcester, MA 01610, U.S.A. :~Abbreviations used: AAF, N-2-acetylaminofluorene; AF, _N-2-aminofluorene; AcO, acetoxy; AAIF, N-2acetylamino-7-iodofluorene. 0022-2836/90/100239-08 $03.00/0
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© 1990 Academic Press Limited
240
G. R. Hoffmann and R. P. P. Fuchs
ft,/-
Insertion- denaturation model
N-AcO-AAIF
Outside bindinG model
Figure 1. Models for the effects of AAF adducts and AAIF adducts on the structure of DNA (Fuchs et al., 1976). displacement model, for the binding of AAF to DNA (Daune et al., 1981; Fuchs et al., 1976; Grunberger et al., 1970). According to this model, guanine residues modified by --AAF rotate from the anti conformation to the syn conformation and are shifted outside the double helix; the fluorene ring is inserted into the DNA molecule in place of the guanine moiety, resulting in a local disorganization of the DNA structure (Daune et al., 1981; Fuchs, 1975; Fuchs & Daune, 1972, 1973, 1974; Fuchs et al., 1976; Lang et al., 1979; Lef~vre et al., 1978; Sage et al., 1979). The binding of AF to DNA is not as well characterized as that of AAF (Duane et al., 1981) but it has been described as either insertion without denaturation or outside binding in the major groove of DNA (Daune et al., 1981; Fuchs et al., 1976; Lef~vre et al., 1978). Lacking the acetyl group, AF a~lducts can apparently be accommodated without a major alteration in DNA structure. The spectra of mutations induced by AAF adducts (Koffel-Schwartz et al., 1984) and AF adducts (Bichara & Fuchs, 1985) have been characterized by sequencing forward mutations in the tetracycline resistance gene of plasmid pBR322 in Escherichia coli (Fuchs et al., 1981, 1983, 1988). Although both AAF and AF induce targeted mutations as a consequence of adducts at the C-8 position of guanine, they differ markedly from one another in mutation spectrum. More than 90~/o of the mutations induced by AAF are frameshift
mutations (Koffel-Schwartz et al., 1984), whereas the majority of the mutations induced by AF are base-pair substitutions, primarily G ' C to T-A transversions (Bichara & Fuchs, 1985). The distribution of AAF-induced mutations within the gene is highly non-random, to the extent that 89~o of the mutations can be ascribed to 19~/o of the AAF adducts (Fuchs, 1983; Koffel-Schwartz et al., 1984). The hotspots of mutagenesis by AAF are of two kinds: repetitive sequences and specific non-repetitive sequences, especially the N a r I restriction enzyme recognition sequence GGCGCC (KoffelSchwartz et al., 1984). In contrast, the mutations induced by AF seem to be randomly distributed among guanine residues (Bichara & Fuchs, 1985). The comparison between AAF and AF reveals that related aromatic amines that differ in conformational changes that they cause in DNA (Daune et al., 1981) also differ in mutagenicity (Bichara & Fuchs, 1985; Koffel-Schwartz et al., 1984). Because of such differences, the aromatic amines are useful model compounds for exploring relationships between physical changes induced by the binding of chemicals to DNA and mutagenic effects. The 7-iodo derivative of AAF, N-2-acetylamino-7-iodofluorene (AAIF) is of interest because it differs sharply from AAF with respect to conformational changes that its adducts cause in DNA. The effects of AAIF adducts and AAF adducts on the structure of DNA are illustrated in Figure 1. Just as the principal adduct formed by the reaction of N-AcOAAF with DNA nucleotides is N-(guanin-8-yl)-2AAF, the principal adduct formed in the reaction of N-AcO-AAIF is N-(guanin-8-yl)-2-AAIF (Lef~vre et al., 1978). On the basis of thermal stability, pattern of digestion by endonuclease S~, formaldehyde unwinding, linear dichroism and circular dichroism, it has been proposed that the interaction of AAIF with DNA can be described by an outside binding model (Fuchs & Daune, 1973; Fuchs et al., 1976; Lang et al., 1979; Lefevre et al., 1978), unlike the insertion-denaturation caused by AAF. It seems that steric hindrance associated with the bulky iodo substituent prevents insertion, thereby keeping the fluorene ring outside in the groove of DNA. The guanine residue to which the AAIF is bound at the C-8 position remains stacked with adjacent bases (Daune et al., 1981). Although there are clear differences between AAIF adducts and AAF adducts, there are also such similarities as an ability to stimulate the unwinding of supercoiled plasmid DNA (Lang et al., 1979). Nevertheless, the bulk of evidence indicates that, relative to AAF adducts, both AAIF adducts and AF adducts cause less severe conformational changes in DNA, though for different reasons. Comparing the mutagenicity of AAIF with that of AAF and AF can therefore elucidate relationships between alterations of DNA conformation and genetic effects. AAIF has been shown to induce reversion of the hisD3052 allele in Salmonella typhimurium strain TA98 (Santella et al., 1979). It also induces rever-
A A I F Mutation Spectrum sion in plasmid p X 2 (Fuchs & Bintz, 1990), a pBR322 derivative (Burnouf & Fuchs, 1985) t h a t has a GC dinucleotide inserted at position 435 of the tetracycline resistance gene. Although these results indicate t h a t A A I F induces frameshift mutations, they do not allow a comparison of A A I F to A A F and A F with respect to m u t a t i o n a l mechanisms because of the lack of information on base-pair substitutions and frameshift m u t a t i o n s in various sequence contexts. I f the genetic effects of A A F are, in fact, relatable to the structural alterations t h a t this c o m p o u n d causes in DNA, then one m i g h t expect the m u t a t i o n s p e c t r u m of A A I F to differ from t h a t of A A F and to show some similarity to t h a t of AF.
2. Materials and Methods (a) Bacteria and plasmids All experiments were conducted with plasmid pBR322 (Bolivar et al., 1977) and E. cell strain ABll57 (thr-1 ara-14 leuB6 A(gpt-proA)62 lacY1 tsx-33 supE44 talK2 ,~- rac- hisG4(Oc) rfbD1 mgl-51 rpsL31 kdgK51 xyl-5 mtl-1 argE3 thi-l: Bachmann, 1987). The bacteria were grown in LB medium (Maniatis et al., 1982). Plasmid pBR322 is a multicopy plasmid carrying genes that confer on its host resistance to tetracycline and ampicillin. The plasmid was grown in strain ABl157 and isolated as described (Fuchs et al., 1981; 1988).
241
base-pair segment that serves as the target for mutagenesis in the forward mutation assay. Untreated pBR322 and the modified DNA samples containing about 40 or 100 AAYF adducts/plasmid were subjected to digestion by BamHI and SalI. The target fragment (6 S) was isolated from the remainder (16 S) of the plasmid by sucrose gradient centrifugation. The 6 S fragments containing 0, 2-5 or 6"5 AAIF adducts were ligated by unmodified 16 S fragments using phage T4 ligase as described by Fuchs et al. (1988). The AAIF-induced damage in the reconstructed plasmids was therefore confined to the 276 basepair target sequence. Cells of E. coil strain ABll57 (A~oo -- 0-2) were centrifuged, suspended in the same volume of 10 mM-MgS04, and irradiated with ultraviolet light (approx. 30 J/m 2) from a Philips 15 W germicidal lamp to induce SOS functions. The cells were grown in LB medium for 30 min to allow expression of SOS functions before transformation. Piasmids containing AAIF adducts in the target sequence were introduced into the cells by CaCl2-transformation as described by Fuchs ctal. (1988). Transformants were selected on LB plates containing ampicillin (50 gg/ml). Single colonies were tested for tetracycline sensitivity on LB plates with and without tetracycline (0, 20 and 40 pg/ml). Mutants were identified on the basis of their resistance to ampicillin but sensitivity to tetracycline (40/~g/mi). Clones that grew in the presence of tetracycline at 20 pg/ml but not at 40/~g/ml were considered to be leaky mutants.
(e) Sequence analysis of mutations 4
(b) Chemicals The 7-iodo derivative of N-AcO-AAF (N-AcO-AAIF) was synthesized as described (Lef~vre et al., 1978) and purified by high-pressure liquid chromotography; its purity was confirmed by ultraviolet light spectrophotometry. Enzymes were purchased from New England Biolabs, Beverly, MA, U.S.A., and used as specified by the manufacturer. Tetracycline and ampicillin were purchased from Serva, Heidelberg, F.R.G. All chemicals were reagent grade. (c) Modification of pBR322 by N-AcO-AAIF Plasmid DNA (50 pg/100 pl) was modified in vitro by reacting it at 37°C with N-AcO-AAIF ( ~ 0"3 ~g/ml) in 2x 10 -5 M-citrate buffer (pH 7), containing 5~/o (v/v) ethanol. The modified DNA was extracted 3 times in chloroform and precipitated in ethanol saturated with sodium acetate as recommended by Fuchs et al. {1988). AAIF adducts in the DNA were quantified by ultraviolet light spectrophotometry on the basis of the AAIF absorbance peak at 310 nm, given the extinction coefficient of 23,000 (Lef~vre et al., 1978). The extents of modification in 2 DNA samples were estimated as 40"6 adducts/plasmid (A31o/A260 = 0"0167) and 104 adducts/plasmid (A3to/A26 o = 0"0429) after reaction times of about 15 rain and 45 min, respectively. (d) Mutation assay The spectrum of mutations induced by AAIF was studied in a forward mutation assay based on inactivation of the tetracycline resistance gene of pBR322 (Fuchs et al., 1981, 1983, 1988). This gene is 1188 base-pairs long and has BamHI and SalI restriction sites at positions 375 and 651, respectively. The 2 restriction sites therefore delimit a 276
A rapid boiling mini-prep procedure (Holmes & Quigley, 1981) was used to isolate plasmid DNA from 30 tetracycline-sensitive clones recovered from SOS-induced cells transformed with pBR322 containing about 6"5 AAIF adducts/6 S fragment (i.e. equivalent to about 100 adducts/plasmid). Plasmids that migrated faster than wild-type pBR322 in agarose-gel electrophoresis (1/30) or were not susceptible to digestion by BamHI and SalI (2/30) were eliminated from further analysis. Plasmid DNA for mutational analysis was isolated and purified by cesium chloride density gradient centrifugation. A I0 gg sample of the DNA was digested by BamHI and SalI, dephosphorylated with calf intestinal alkaline phosphatase, and end-labeled with [32P]ATP and T4 polynucleotide kinase. Single strands of the 6 S BamHI-SalI fragment were isolated by acrylamide gel electrophoresis, eluted from the gel, and precipitated in ethanol. The nueleotide sequences of the 6 S fragments were determined by Maxam & Gilbert chemistry (Maxam & Gilbert, 1977, 1980) followed by electrophoresis on 8% and 20% (w/v) acrylamide sequencing gels. Details of the materials and methods used in the forward mutation assay have been reviewed (Fuchs et al., 1988).
3. Results (a) Transformation and recovery of mutants
E. cell strain AB1157 was t r a n s f o r m e d with plasmids t h a t had been reconstructed b y ligating 6 S f r a g m e n t s containing 0, 2-5 or 6"5 A A I F a d d u c t s to the remainder of the plasmid f r o m unmodified pBR322. T r a n s f o r m a t i o n efficiencies were e s t i m a t e d from n u m b e r s of t r a n s f o r m a n t s per n a n o g r a m of t r a n s f o r m i n g D N A relative to control t r a n s f o r m a tions, m a k i n g the a s s u m p t i o n t h a t the efficiency of
G. R. Hoffmann and R. P. P. Fuche
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Table 1
Induction of mutations by A A I F in the tetracycline-resistance gene of plasmid pBR322 AAIF adduets per 6 S
Bam-Sal
Plasmid
fragment
pBR322 control 16 S+6 S ligated 16 S+6 S ligated 16 S+6 S ligated 16 S+6 S ligated
SOS induction of host
Transforming efficiencyt
Relative transforming efficiency:~
Clones tested
Mutants
Mutant frequency (%)
_ + + + -
1"000 0'036 0.026 0"017 0"009
-1'00 0"72 0"47 --
31 315 865 1366 617
0 0 4 30 2
-0'00 0"46 2-20 0'32
0 0 2-5 6"5 6"5
t Relative to the pBR322 control. :~Relative to the unmodified ligated plasmid.
l i g a t i o n w a s t h e s a m e in t h e t h r e e s a m p l e s . T h e d a t a a r e p r e s e n t e d in T a b l e 1. T h e d e c r e a s e d t r a n s f o r m i n g efficiencies w i t h 2"5 or 6"5 A A I F a d d u c t s p e r p l a s m i d reflect t h e r e d u c t i o n o f v i a b i l i t y o f plasmids bearing these adducts. T o d e t e c t f o r w a r d m u t a t i o n s i n d u c e d b y A A I F in t h e t a r g e t 6 S BamHI-SalI r e s t r i c t i o n f r a g m e n t , i n d i v i d u a l clones w e r e t e s t e d for s e n s i t i v i t y t o t e t r a c y c l i n e (40 p g / m l ) . N o s p o n t a n e o u s t e t r a c y c l i n e s e n s i t i v e m u t a t i o n s w e r e d e t e c t e d in t h e s m a l l s a m p l e (315 clones) o f t r a n s f o r m a n t s f r o m u n m o d i fied p B R 3 2 2 t h a t w a s t e s t e d , b u t h i s t o r i a l c o n t r o l
d a t a ( F u c h s et al., 1988) i n d i c a t e t h a t t h e f r e q u e n c y o f s p o n t a n e o u s m u t a t i o n s in S O S - i n d u c e d cells is a b o u t 6 x l 0 -4. T h e d a t a in T a b l e 1 s h o w a dosed e p e n d e n t i n c r e a s e in m u t a t i o n f r e q u e n c i e s in p l a s m i d s m o d i f i e d w i t h A A I F t o t h e e x t e n t o f 2"5 a n d 6"5 a d d u c t s p e r 6 S f r a g m e n t . T h e p r o p o r t i o n s o f l e a k y m u t a n t s , d e f i n e d b y g r o w t h on t e t r a c y c l i n e a t 20 p g / m l b u t n o t a t 40 p g / m l , w e r e 1 o f 4 a t t h e l o w e r dose a n d 4 o f 30 a t t h e h i g h e r dose. T h e f r e q u e n c y o f m u t a n t s r e c o v e r e d a t t h e h i g h e r dose is a b o u t 35-fold t h e e x p e c t e d s p o n t a n e o u s f r e q u e n c y . The m u t a t i o n experiments were conducted with
Table 2
Mutations induced by A A I F in the B a m - S a l fragment of the tetracycline-resistance gene of plasmid pBR322 Position in the Tc' genet
Mutation number
Mutation:[: Description of the site of the mutation
390 400-401 402-403 419-420
5-53 13-103 11-87 16-28b
G--*C -G --C +G
420 428 439-440
5-111 6-82 16-28a
C--*G G--*T -CT
461 474 520-521
1-108~ 6-125 6-61
G--*T C--*A -G
528 530 532-533 536 537 536-540 536-540 536-540 537-540 549-552 573 573-574
6-63 1-100~ 5-59 14-112a 6-31 11-120 1-42 12-101 14-112b 1-67 4-113§ 7-26
C-~G G-~C - G G-*T G-*T +G -G -G -G -GC T --*C +C
586-587
4-76
-G
613-615
1-56
-G
GG dinucleotide in the sequence CCG_GAC GG dinucleotide in the sequence GTG_GGCC CC dinueleotide in the sequence GGCCGG G insert between repetitive CA dinucleotides in the sequence CACA In repetitive CA dinucleotides of the sequence CACA GG dinucleotide in the sequence CGGTT Loss of dinucleotide from sequence C-C__T_TAadjacent to a NarI site Run of 4 G residues Adjacent to run of 3 G residues GG dinucleotide in the sequence TGGTGG; a hotspot for mutagenesis by AAF Run of 4 C residues Adjacent to run of 4 C residues GG dinucleotide in the sequence TG__GC Run of 5 G residues Run of 5 G residues Run of 5 G residues Run of 5 G residues Run of 5 G residues Run of 5 G residues NarI site (GGCGCC); a hotspot of mutagenesis by AAF TT dinucleotide in the sequence ATTCC C insert between TT and CC dinucleotides in the sequence TTCC GG dinucleotide adjacent to a run of trinucleotides: GCGGCGGCGG Run of 3 G residues
t Numbering as defined by Sutcliffe (1979). :~All mutations are described for the DNA strand whose sequence corresponds to that of mRNA. § Phenotypieally leaky (grows at 20 #g tetracycline/ml but not at 40 pg tetracyeline/ml).
Coding change Gly~Ala - 1 frameshift - 1 frameshift + 1 frameshift Thr--*Arg Val-*Phe - 2 frameshift Gly-*Trp Ala-~Asp - I frameshift Pro--*Arg Val-*Leu - 1 frameshift Gly--*Trp Gly-*Val + l frameshift - 1 frameshift - 1 framesbift - 1 frameshift - 2 frameshift Phe --*Ser + 1 frameshift - 1 frameshift - 1 frameshfft
AAIF
Mutation Spectrum
243
Table 3
Comparison of the mutation spectrum of A A I F to the spectra of A A F and A F AAIF
AAF~f
AF:~
A. Classification of the mutations (l) Frameshift mutations + 1 frameshifts 1 frameshifts 2 frameshifts 3 deletions~ (2) Base-pair substitutions Transitions Transversions GC to TA GC to CG AT to TA
14/24
32/34 3/14 9/14 2/14 0/14
-
-
-
10/24
6/23 2/32 13/32 12/32 5/32
2/34 1/ 10 9] 10 5/9 4]9 0/9
0/6 4/6 2/6 0/6 17/23
1/2 1/2 0/1 0/1 1/1
1/ 17 16/17 14/16 1/16 1/16
B. Sites of the mutations (1) Mutations initiated at a G-C base-pair (2) Mutations initiated at an A ' T base-pair
23/24 1/24
33/34 1/34
22/23 1/23
0/24 1/24 23/24
0/34 12/34 22/34
4/23 2/23 17/23
C. Sequences surroundinq the sites of the mutations (1) Mutations in non-repetitive, sites (2) - 2 frameshffts in NarI'sites (GGCGCC) (3) Mutations associated with a repetitive site (i) Extent of repetition: Run of 5 bases Run of 4 bases Run of 3 bases Run of 2 bases Repetitive dinucleotides (e.g. CACA) Repetitive trinucleotide (--3 in GCGGCGGCG) (ii) Association with the repetition: Frameshifts at repetitive sites Proportion of frameshffts at repetitive sites Proportion of frameshifts in runs of 3, 4 or 5 bp Deletion of repetitive trinucleotide Base-pair substitutions at repetitive sites Proportion of substitutions at repetitive sites Proportion of substitutions in runs of 3, 4 or 5 bp Mutations immediately adjacent to a run of 3, 4 or 5 bp Insertion, deletion or substitution between two repeats (e.g. TTXCC)
6/23 3/23 2/23 10/23 2/23 0/23 12/23
2/21 4/21 1/21 9/21 1/21 5/21 15/21
12/14 5/12 0/23 8/23
1/17 1/17 7/17 8/17 0/17 0/17 3/17
15/32 7/15 5/21 2/21
8/10 4/8
3/6 1/3 0/17 11/17
2/2 0/1
11/17 7/11
2/23 1/23
0/21 0/21
1/17 2/17
20/22 2/22
34/36 2/36
18/20 2]20
D. Occurrenceof complex or multiple mutationsll (1) Simple mutations (2) Complex or multiple mutations
J" Based on Koffel-Schwartz et al. (1984); includes data from wild-type and uvrA strains of E. coll. bp, base-pairs. J: Based on Bichara & Fuchs (1985). § Though not frameshifts in a strict sense, --3 mutations are pooled with frameshifts because they involve deletions of base-pairs (bp). ]1The multiple (or complex) mutations induced by AAIF and AF are included in the Table; the total numbers of mutations are therefore 24 and 23, respectively. The complex mutations induced by AAF are omitted because they are not readily described as a combination of simple mutations.
cells w h o s e S O S s y s t e m h a d b e e n i n d u c e d b y u l t r a v i o l e t l i g h t , b e c a u s e m u t a g e n e s i s b y A A F (KoffelS c h w a r t z et al., 1984) a n d A F ( B i c h a r a & F u c h s , 1985) is k n o w n t o be d e p e n d e n t on S O S f u n c t i o n s o f t h e host. T r a n s f o r m a t i o n i n t o u n i n d u c e d cells revealed that mutagenesis by AAIF similarly d e p e n d s on h o s t S O S f u n c t i o n s . T h e d e p e n d e n c e , m o r e o v e r , m a y b e g r e a t e r t h a n t h e s e v e n f o l d difference s h o w n in T a b l e 1, b e c a u s e t h e p u t a t i v e m u t a n t s r e c o v e r e d in t h e S O S - i n d u c e d cells w e r e confirmed by subsequent analysis, while the two m u t a n t s r e c o v e r e d in t h e u n i n d u c e d cells w e r e n o t analyzed further.
(b) Sequence analysis of mutations induced by A A I F S e q u e n c e a n a l y s i s w a s p e r f o r m e d o n 22 m u t a n t s from plasmids that had been modified with AAIF to t h e e x t e n t o f 6"5 a d d u c t s p e r B a m t t I - S a l I f r a g m e n t . T h e m u t a t i o n s a r e l i s t e d in T a b l e 2. T h e T a b l e s h o w s 24 m u t a t i o n s b e c a u s e p l a s m i d s 1 4 - 1 1 2 a n d 16-28 turned out to be double mutants. The fact t h a t all t h e m u t a t i o n s e x c e p t o n e a r e i n i t i a t e d a t a G-C base-pair suggests that the mutations are t a r g e t e d a t s i t e s o f A A I F a d d u c t s on g u a n i n e residues. Table 2 reveals that the majority of AAIF-
244
G. R. Hoffmann and R. P. P. Fuchs
induced mutations are transversions and frameshift mutations involving the loss of a single base-pair. Frameshift mutations involving the loss of two base-pairs or the gain of a single base-pair were also observed. The data also reveal that the mutations are strongly associated with repetitive sequences, including a prominent hotspot at position 536 to 540, which is a run of five G'C base-pairs. The classes of mutations that comprise the mutation spectrum of AAIF are summarized and compared to the mutation spectra of AAF and AF in Table 3. The most conspicuous difference among the mutation spectra is the relative proportion of frameshift mutations and base-pair substitutions. The ratio of substitutions to frameshifts was l0 : 14 forAAIF, 2 : 3 2 for AAF (Koffel-Schwartz et al., 1984), and 17 : 6 for AF (Bichara & Fuchs, 1985). Contingency X2 calculations show the distribution of substitutions and frameshifts induced by AAIF to differ significantly from that induced by either AAF or AF. 4. Discussion
The frequencies of mutations induced by AAIF in SOS-induced cells (Table 1) correspond to 0"0018 mutation per adduct in plasmids carrying 2"5 adducts per 6 S fragment and 0"0034 mutation per adduct in plasmids with 6"5 adducts per 6 S fragment. The difference between the two dosages suggests that the efficiency with which AAIF adducts are processed into mutations is higher when more adducts are present. These mutagenic efficiencies are similar to those reported for AAF, where there were 0"0019 and 0"0025 mutation per adduct at 3 and 6 adducts per 6 S fragment, respectively (Koffel-Schwartz et al., 1984). Although AAIF adducts are comparable to AAF adducts in mutagenic potency, they differ from AAF adducts with regard to the kinds of mutations induced. Whereas AAF induces frameshift mutations almost exclusively (Koffel-Schwartz et al., 1984), AAIF induces frameshift mutations and base-pair substitutions in roughly equal numbers. It is likely that the proportion of base-pair substitutions shown in Table 3, like that in any assay that detects mutations on the basis of phenotype, is underestimated because some base-pair substitutions may be silent or leaky enough to preclude detection. Nevertheless, the observation that about 50o/o of AF-induced mutations are phenotypically leaky (Bichara & Fuchs, 1985), but only about 15% of AAIF-induced mutations are leaky by the same criterion, is consistent with the sequencing data (Bichara & Fuchs, 1985) in indicating that AF is more specific than AAIF as a base-pair substitution mutagen. The comparative data therefore place the mutation spectrum of AAIF intermediate between those of AAF and AF. Rather than simply being intermediate, the mutation spectrum of AAIF has properties that differ from both AAF and AF. Conclusions in this respect are somewhat tenuous because of limited
numbers of mutations sequenced, and additional differences or spectral complexities might be revealed if the analysis for all three compounds were enlarged. Nevertheless, certain trends are evident from a comparison of the mutation spectra summarized in Table 3. Besides containing more frameshift mutations, the mutation spectrum of AAIF differs from that of AF in the proportion of different classes of base-pair substitutions. Both agents induce transitions only rarely; however, the spectrum of AAIF includes G" C to T ' A transversions and G'C to C'G transversions in equal numbers, while that of AF includes G" C to T" A transversions almost exclusively. Similarly, the distribution of frameshift mutations induced by AAIF differs from that induced by AAF. The frameshift mutations induced by AAIF are principally - 1 (9/14) and + 1 (3/14) frameshifts, and they are strongly associated with repetitive guanine residues. Repetitive sequences are also hotspots for mutagenesis by AAF (Koffel-Schwartz et al., 1984); however, the spectrum of frameshift mutations induced by AAF includes a high frequency of - 2 frameshifts in N a r I sites (GGCGCC) (Koffel-Schwartz et al., 1984). Only one such mutation was found among 24 AAIF-induced mutations. Multiple mutations in the tetracycline resistance gene seem to occur at a frequency higher than that expected for independent events. In the current study, mutant 16-28 contains a + 1 frameshift at position 419 to 420 and a - 2 frameshift at position 439 to 440. Mutant 14-112 has a G ' C to T ' A transversion at position 536 and a - 1 frameshift at position 537 to 540. In view of the proximity of the sites, it seems likely that mutant 14-112 represents a single complex mutation in which the two central guanine residues in the sequence CCGGGG were replaced by a thymine. Complex mutations and multiple mutations have been observed repeatedly in the forward mutation assay in the tetracycline resistance gene: two induced by AAIF, two by AAF (Koffel-Schwartz et al., 1984), two by AF (Bichara & Fuchs, 1985) and four by cisplatin (Burnouf et al., 1987); in each case, the sample sizes and mutation frequencies would support a prediction of about 0"5 multiple mutation under the assumption that the mutations are independent events. The mechanisms underlying the complex and multiple mutations are obscure. Some of the differences in mutation spectra of AAF, AF and AAIF can be interpreted with respect to conformational changes that the compounds induce in DNA. AAF adducts cause a major conformational change, described by an insertiondenaturation model (Daune et al., 1981). In contrast, AF causes a less severe conformational change that may be ascribed to insertion without denaturation or outside binding (Bichara & Fuchs, 1985; Daune et al., 1981). Analyses of DNA structure (Fuchs & Daune, 1973; Fuchs et al., 1976; Lang et al., 1979; Lef6vre et al., 1978) suggest that AAIF does not cause the severe distortion associated with AAF, presumably because the iodo group sterically
AAIF
245
Mutation Spectrum
interferes with insertion. As might be expected from the structural data, the analysis of mutations induced by AAIF reveals a mutation spectrum that resembles that of AAF in some respects and AF in others. Several lines of evidence suggest that the - 2 frameshift mutations induced by AAF in NarI sites (G1G2CG3CC) are associated with destabilization of the B structure of DNA in the vicinity of AAF adducts on the third guanine residue, forming a local Z-like structure (Koffel-Schwartz et al., 1984; Burnouf et al., 1989; Koehl et al., 1989). In contrast to AAF adducts, AAIF adducts are less effective in inducing - 2 frameshift mutations in N a r I sites. The explanation for this difference between the two mutagens may lie in the differences in the conformational changes that they cause. The AAF adduct causes the guanine to rotate from the anti to the syn conformation (Daune et al., 1981; Evans et al., 1980). This conformational change may trigger the B to Z transition in stretches of alternating GC residues; elsewhere, it may cause the local denaturation associated with insertion of the fluorene ring and extrusion of the guanine. In contrast, the rotation of guanine from anti to syn seems to be sterically blocked in the case of AAIF adducts (Lef~vre et al., 1979). Consequently, AAIF adducts should be less effective than AAF adducts in stimulating the B to Z transition that appears to be centrally involved in the N a r I mutation pathway. The degree to which repetitive sequences are hotspots seems to depend on the length of the repeat; this observation is consistent with mutation spectra obtained in other systems (Ripley et al., 1986). The longest repeat in the target fragment used in our study is the run of five guanine residues at site 536 to 540. This single run accounts for one-fourth of the mutations induced by AAIF. Correcting for the number of occurrences of runs of different lengths, one observes t h a t the run of five guanine residues is more mutable than runs of four, and runs of four guanine residues are more mutable than those of two or three. Eveh runs of two or three guanine residues, however, are more mutable than isolated guanine residues. The slippage model (Streisinger & Owen, 1985; Strei§inger et al., 1966) can explain the high susceptibility of repetitive sequences to frameshift mutagenesis by AAIF. The data in Table 2, however, suggest that repetitive sequences are hotspots not only for frameshift mutations but also for ,transversions induced by AAIF. A tendency for transversions to be more frequent in runs than in nonrepetitive sequences can also be discerned among the mutations induced by AF in the same assay (Bichara & Fuchs, 1985; Table 3). Recent evidence suggests that a template dislocation model c a n explain the occurrence of some transversions at sites of repetitive bases (Boosalis et al., 1989; Kunkel & Alexander, 1986). According to this model, a transient misalignment during replication leads to the misincorporation of a base at the site of the 5'-most base in the template strand of a short run; the new
base is complementary to the next base in the template. Although the model has been considered principally for hotspots of spontaneous mutagenesis, it could also be applicable to induced mutations. Inspection of the sequences surrounding the transversions induced by AAIF adducts reveals that only mutation 1-108 clearly conforms to the model. One may speculate therefore t h a t other factors must contribute to the tendency for transversions to occur preferentially at sites of repetitive bases. In this respect, it has recently been shown (Seeberg & Fuchs, 1990) that AAF adducts on the three guanine residues in the sequence 5'-GtG2CGaCC-3' are not equally susceptible to incision by the u v r A B C excinuclease. Although the result may be specific to this particular sequence, the reduced incision when AAF adducts are on G2 suggests the possibility that adducts on guanine residues may be less efficiently repaired in general when the adjoining 5' nucleotide is also a guanine. I f so, it is conceivable that the difference in reparability of adducts in runs of guanine residues or in GG dinucleotides may also contribute to these sequences being hotspots for mutagenesis by AAIF. We thank Professor Michel Daune whose insight into the structural effects of AAIF provided the impetus for studying its mutagenicity. We also thank Marc Bichara, R~gine Bmtz, Dominique Burnouf, Anne-Marm Freund, Patrice Koehl and Xavier Veaute for valuable discussions and collaboration during the course of this study and Mme Liliane Diebolt for excellent secretarial work on the manuscript. This work was supported partly by grants from the Association pour la Recherche sur le Cancer (no. 6143), from the F~d~ration Nationale des Centres de Lutte contre le Cancer and from the MRES "action sondes froides." The support of a fellowship from the NIH French CNRS Program for Scientific Collaboration and sabbatical support from the College of the Holy Cross, Worcester, MA, U.S.A., are gratefully acknowledged• •
e
,
.
References Bachmann, B. (1987) In Escherichia coil and Salmonella typhimurium: Cellular and Molecular Biology (Ingraham, J.L., Low, K.B., Magasanik, B., Schaechter, M. & Umbarger, H.E., eds), pp. 1190-1219, Amer. Soc. Microbiol., Washington, D.C. Bichara, M. & Fuchs, R. P. P. (1985). J. Mol. Biol. 183, 341-351. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L., Boyer, H. W., Crosa, J. H. & Falkow, S. (1977). Gene, 2, 95-113. Boosalis, M. S., Mosbaugh, D. W., Hamatake, R., Sugino, A., Kunkel., T. A. & Goodman, M. F. (1989). J. Biol. Chem. 264, 11360-11366. Burnouf, D. & Fuehs, R. P. P. (1985). Biochimie, 67, 385-389. Burnouf, D., Daune, M. & Fuehs, R. P. P. (1987). Proc. Nat. Acad. Sci., U.S.A. 84, 3758-3762. Burnouf, D., Koehl, P. & Fuchs, l~. P. P. (1989). Proc. Nat. Acad. Sci., U.S.A. 86, 4147-4151. Daune, M. P., Fuchs, R. P. & Leng, M. (1981). Nat. Cancer Inst. Monogr• 58, 201-210.
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Evans, F. E., Miller, D. W. & Beland, F. A. (1980). Carcinogenesis, 1,955-959. Fuchs, R. P. P. (1975). Nature (London), 257, 151-152. Fuchs, R. P. P. (1983). J. Mol. Biol. 177, 173-180. Fuchs, R. P. P. & Bintz, 1%. (1990). Environmental Health Perspectives, in the press. Fuchs, 1%. & Daune, M. (1972}. Biochemistry, 11, 2659-2666. Fuchs, 1%. & Daune, M. (1973). FEBS Letters, 34, 295-298. Fuchs, R. P. & Daune, M. (1974). Biochemistry, 13, 4435-4440. Fuchs, R. P. P., Lef~vre, J.-F., Pouyet, J. & Daune, M. P. (1976). Biochemistry, 15, 3347-3351. Fuchs, 1%. P. P., Schwartz, N. & Daune, M. P. (1981). Nature (London), 294, 657-659. Fuchs, R. P. P., Koffel-Schwartz, N. & Daune, M. (1983). In Cellular Responses to DNA Damage (Friedberg, E. C., ed.), pp. 547-557, Alan 1%. Liss, New York. Fuchs, R. P. P., Bichara, M., Burnouf, D. & KoffelSchwartz, N. (1988). In DNA Repair: A Laboratory Manual of Research Procedures (Friedberg, E. & Hanawalt, P., eds), vol. 3, pp. 235-257, Marcel Dekker, New York. Grunberger, D., Nelson, J. H., Cantor, C. R. & Weinstein, I.B. (1970). Proc. Nat. Acad. Sci., U.S.A. 66, 488-494. Holmes, D. S. & Quigley, M. (1981). Anal. Biochem. 114, 193-197. Koehl, P., Valladier, P., Lef~vre, J.-F. & Fuchs, R. P. P. (1989). Nucl. Acids Res. 17, 9531-9541. Koffel-Schwartz, N., Verdier, J.-M., Bichara, M. ]~reund, A.-M., Daune, M.P. & Fuchs, R . P . P . (1984). J. Mol. Biol. 177, 33-51. Kriek, E. (1965}. Biochem. Biophys. Res. Commun. 2{}, 793-799.
Kriek, E., Miller, J. A., Juhl, U. & Miller E. C. (1967). Biochemistry, 6, 177-182. Kunkel, T. A. & Alexander, P. S. (1986). J. Biol. Chem. 261, 160-166. Lang, M. C. E., Freund, A.-M., de Murcia, G., Fuchs, R. P. P. & Daune, M. P. (1979). Chem.-Biol. Interact. 28, 171-180. Lef'evre, J.-F., Fuchs, R. P. P. & Daune, M. P. (1978). Biochemistry, 17, 2561-2567. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Maxam, A. M. & Gilbert, W. (1977). Proc. Nat. Acad. Sci., U.S.A. 74, 560-564. Maxam, A. M. & Gilbert, W. (1980). Methods Enzymol. 65, 499-560. Ripley, L. S., Clark, A. & deBoer, J. G. (1986). J. Mol. Biol. 191,601-613. Sage, E., Spodheim-Maurizot, M., Rio, P., Leng, M. & Fuchs, 1%. P. P. (1979). F E B S Letters, 108, 66-68. Santella, R. M., Fuchs, 1%. P. P. & Grunberger, D. (1979). Mutat. Res. 67, 85-87. Seeberg, E. & Fuchs, R. P. P. (1990). Proc. Nat. Acad. Sei., U.S.A. 87, 191-194. Streisinger, G. & Owen, J. (1985). Genetics, 109, 633-659. Streisinger, G., Okada, Y., Emrich, J., .Newton, J., Tsugita, A., Terzaghi, E. & Inouye, M. (1966). Cold Spring Harbor Syrup. Quant. Biol. 31, 77-84. Sutcliffe, J. G. (1979). Cold Spring Harbor Syrup. Quant. Biol. 43, 77-90. Tang, M. & Lieberman, M. W. (1983). Carcinogenesis, 4, 1001-1006. Westra, J. G. & Visser, A. (1979). Cancer Letters, 8, 155-162.
Edited by P. Chambon
D N A Sequence Analysis of Mutations Induced by N-2-Acetylamino-7-iodofluorene in Plasmid pBR322 in Escherichia coli George R. Hoffmann~f and Robert P. P. Fuchs Groupe de Cancdrogdn~se et de Mutagdn~se Moldculaire et Structurale Institut de Biologic Moldculaire et Cellulaire Centre National de la Recherche Scientifique 15 rue Rend Descartes, 67084 Strasbourg cedex, France (Received 11 September 1989; accepted 19 January 1990) The spectrum of mutations induced by N-2-acetylamino-7-iodofluorene (AAIF) was analyzed in a forward mutation system based on mutagenesis directed to a small restriction fragment in the tetracycline resistance gene of plasmid pBR322. AAIF was found to induce frameshift mutations and base-pair substitutions at approximately equal frequencies. The frameshift mutations were mostly deletions of single base-pairs, but - 2 frameshifts and + 1 frameshifts were also detected. With one exception, the substitutions were transversions initiated at a G ' C base-pair. Both frameshift mutations and transversions occurred preferentially at sites of repetitive guanine residues. Although AAIF and the related aromatic amines N-2-acetylaminofluorene (AAF) and N-~-aminofluorene (AF) all bind to the C-8 position of guanine, they have different effects on DNA conformation, and these differences are reflected in their mutation spectra. Previous studies have provided evidence that AAF adducts can trigger a B to Z conformational change in alternating GC sequences or displacement of the guanine by the fluorene ring in other sequences; the principal result is two classes of frameshift mutations. AF, whose DNA interaction involves outside binding rather than insertion and denaturation, primarily induces base-pair substitutions. AAIF adducts are chemically similar to AAF adducts, but the iodo group apparently hinders insertion of the fluorene ring into DNA. Consistent with this model, the mutation spectrum of AAIF combines properties of the mutation spectra of both AAF and AF.
nogen N-acetoxy-AAF (N-AcO-AAF). Just as DNA can be treated in vitro with N-AcO-AAF to form AAF adducts, treatment of DNA in vitro with N-hydroxy-N-2-aminofluorene (N-OH-AF) specifically forms AF adducts (Tang & Lieberman, 1983). The principal reaction site in DNA treated with N-AcO-AAF or N-OH-AF is the C-8 position of guanine (Evans et al., 1980; Kriek, 1965; Kriek et al., 1967; Westra & Visser, 1979). Despite the similarity in sites of reaction, AAF and AF differ from one another with respect to conformational changes that their adducts cause in DNA (Daune et al., 1981) and the spectrum of mutations that they induce (Bichara & Fuchs, 1985; Koffel-Schwartz et al., 1984). Diverse lines of evidence, including formaldehyde unwinding, pattern of hydrolysis by endonuelease $1, linear dichroism, circular dichroism, reaction with specific antibodies, and thermal stability, support an insertion-denaturation model, also called a base-
1. I n t r o d u c t i o n
The aromatic amines N-2-acetylaminofluorene (AAF$) and N-2-aminofluorene (AF) have been the subject of intensive investigation with respect to chemical and physical interactions with DNA and mutagenic mechanisms (Bichara & Fuchs, 1985; Daune et al., 1981; Koffel-Schwartz et al., 1984). AAF is a hepatic carcinogen whose carcinogenicity requires the metabolic activation of the parent compound to yield electrophilic metabolites. In order to circumvent the need for metabolic activation, many studies of AAF in non-mammalian systems are conducted with the direct-acting carcit Author to whom all correspondence should be addressed, at Department of Biology, College of the Holy Cross, Worcester, MA 01610, U.S.A. :~Abbreviations used: AAF, N-2-acetylaminofluorene; AF, _N-2-aminofluorene; AcO, acetoxy; AAIF, N-2acetylamino-7-iodofluorene. 0022-2836/90/100239-08 $03.00/0
239
© 1990 Academic Press Limited
240
G. R. Hoffmann and R. P. P. Fuchs
ft,/-
Insertion- denaturation model
N-AcO-AAIF
Outside bindinG model
Figure 1. Models for the effects of AAF adducts and AAIF adducts on the structure of DNA (Fuchs et al., 1976). displacement model, for the binding of AAF to DNA (Daune et al., 1981; Fuchs et al., 1976; Grunberger et al., 1970). According to this model, guanine residues modified by --AAF rotate from the anti conformation to the syn conformation and are shifted outside the double helix; the fluorene ring is inserted into the DNA molecule in place of the guanine moiety, resulting in a local disorganization of the DNA structure (Daune et al., 1981; Fuchs, 1975; Fuchs & Daune, 1972, 1973, 1974; Fuchs et al., 1976; Lang et al., 1979; Lef~vre et al., 1978; Sage et al., 1979). The binding of AF to DNA is not as well characterized as that of AAF (Duane et al., 1981) but it has been described as either insertion without denaturation or outside binding in the major groove of DNA (Daune et al., 1981; Fuchs et al., 1976; Lef~vre et al., 1978). Lacking the acetyl group, AF a~lducts can apparently be accommodated without a major alteration in DNA structure. The spectra of mutations induced by AAF adducts (Koffel-Schwartz et al., 1984) and AF adducts (Bichara & Fuchs, 1985) have been characterized by sequencing forward mutations in the tetracycline resistance gene of plasmid pBR322 in Escherichia coli (Fuchs et al., 1981, 1983, 1988). Although both AAF and AF induce targeted mutations as a consequence of adducts at the C-8 position of guanine, they differ markedly from one another in mutation spectrum. More than 90~/o of the mutations induced by AAF are frameshift
mutations (Koffel-Schwartz et al., 1984), whereas the majority of the mutations induced by AF are base-pair substitutions, primarily G ' C to T-A transversions (Bichara & Fuchs, 1985). The distribution of AAF-induced mutations within the gene is highly non-random, to the extent that 89~o of the mutations can be ascribed to 19~/o of the AAF adducts (Fuchs, 1983; Koffel-Schwartz et al., 1984). The hotspots of mutagenesis by AAF are of two kinds: repetitive sequences and specific non-repetitive sequences, especially the N a r I restriction enzyme recognition sequence GGCGCC (KoffelSchwartz et al., 1984). In contrast, the mutations induced by AF seem to be randomly distributed among guanine residues (Bichara & Fuchs, 1985). The comparison between AAF and AF reveals that related aromatic amines that differ in conformational changes that they cause in DNA (Daune et al., 1981) also differ in mutagenicity (Bichara & Fuchs, 1985; Koffel-Schwartz et al., 1984). Because of such differences, the aromatic amines are useful model compounds for exploring relationships between physical changes induced by the binding of chemicals to DNA and mutagenic effects. The 7-iodo derivative of AAF, N-2-acetylamino-7-iodofluorene (AAIF) is of interest because it differs sharply from AAF with respect to conformational changes that its adducts cause in DNA. The effects of AAIF adducts and AAF adducts on the structure of DNA are illustrated in Figure 1. Just as the principal adduct formed by the reaction of N-AcOAAF with DNA nucleotides is N-(guanin-8-yl)-2AAF, the principal adduct formed in the reaction of N-AcO-AAIF is N-(guanin-8-yl)-2-AAIF (Lef~vre et al., 1978). On the basis of thermal stability, pattern of digestion by endonuclease S~, formaldehyde unwinding, linear dichroism and circular dichroism, it has been proposed that the interaction of AAIF with DNA can be described by an outside binding model (Fuchs & Daune, 1973; Fuchs et al., 1976; Lang et al., 1979; Lefevre et al., 1978), unlike the insertion-denaturation caused by AAF. It seems that steric hindrance associated with the bulky iodo substituent prevents insertion, thereby keeping the fluorene ring outside in the groove of DNA. The guanine residue to which the AAIF is bound at the C-8 position remains stacked with adjacent bases (Daune et al., 1981). Although there are clear differences between AAIF adducts and AAF adducts, there are also such similarities as an ability to stimulate the unwinding of supercoiled plasmid DNA (Lang et al., 1979). Nevertheless, the bulk of evidence indicates that, relative to AAF adducts, both AAIF adducts and AF adducts cause less severe conformational changes in DNA, though for different reasons. Comparing the mutagenicity of AAIF with that of AAF and AF can therefore elucidate relationships between alterations of DNA conformation and genetic effects. AAIF has been shown to induce reversion of the hisD3052 allele in Salmonella typhimurium strain TA98 (Santella et al., 1979). It also induces rever-
A A I F Mutation Spectrum sion in plasmid p X 2 (Fuchs & Bintz, 1990), a pBR322 derivative (Burnouf & Fuchs, 1985) t h a t has a GC dinucleotide inserted at position 435 of the tetracycline resistance gene. Although these results indicate t h a t A A I F induces frameshift mutations, they do not allow a comparison of A A I F to A A F and A F with respect to m u t a t i o n a l mechanisms because of the lack of information on base-pair substitutions and frameshift m u t a t i o n s in various sequence contexts. I f the genetic effects of A A F are, in fact, relatable to the structural alterations t h a t this c o m p o u n d causes in DNA, then one m i g h t expect the m u t a t i o n s p e c t r u m of A A I F to differ from t h a t of A A F and to show some similarity to t h a t of AF.
2. Materials and Methods (a) Bacteria and plasmids All experiments were conducted with plasmid pBR322 (Bolivar et al., 1977) and E. cell strain ABll57 (thr-1 ara-14 leuB6 A(gpt-proA)62 lacY1 tsx-33 supE44 talK2 ,~- rac- hisG4(Oc) rfbD1 mgl-51 rpsL31 kdgK51 xyl-5 mtl-1 argE3 thi-l: Bachmann, 1987). The bacteria were grown in LB medium (Maniatis et al., 1982). Plasmid pBR322 is a multicopy plasmid carrying genes that confer on its host resistance to tetracycline and ampicillin. The plasmid was grown in strain ABl157 and isolated as described (Fuchs et al., 1981; 1988).
241
base-pair segment that serves as the target for mutagenesis in the forward mutation assay. Untreated pBR322 and the modified DNA samples containing about 40 or 100 AAYF adducts/plasmid were subjected to digestion by BamHI and SalI. The target fragment (6 S) was isolated from the remainder (16 S) of the plasmid by sucrose gradient centrifugation. The 6 S fragments containing 0, 2-5 or 6"5 AAIF adducts were ligated by unmodified 16 S fragments using phage T4 ligase as described by Fuchs et al. (1988). The AAIF-induced damage in the reconstructed plasmids was therefore confined to the 276 basepair target sequence. Cells of E. coil strain ABll57 (A~oo -- 0-2) were centrifuged, suspended in the same volume of 10 mM-MgS04, and irradiated with ultraviolet light (approx. 30 J/m 2) from a Philips 15 W germicidal lamp to induce SOS functions. The cells were grown in LB medium for 30 min to allow expression of SOS functions before transformation. Piasmids containing AAIF adducts in the target sequence were introduced into the cells by CaCl2-transformation as described by Fuchs ctal. (1988). Transformants were selected on LB plates containing ampicillin (50 gg/ml). Single colonies were tested for tetracycline sensitivity on LB plates with and without tetracycline (0, 20 and 40 pg/ml). Mutants were identified on the basis of their resistance to ampicillin but sensitivity to tetracycline (40/~g/mi). Clones that grew in the presence of tetracycline at 20 pg/ml but not at 40/~g/ml were considered to be leaky mutants.
(e) Sequence analysis of mutations 4
(b) Chemicals The 7-iodo derivative of N-AcO-AAF (N-AcO-AAIF) was synthesized as described (Lef~vre et al., 1978) and purified by high-pressure liquid chromotography; its purity was confirmed by ultraviolet light spectrophotometry. Enzymes were purchased from New England Biolabs, Beverly, MA, U.S.A., and used as specified by the manufacturer. Tetracycline and ampicillin were purchased from Serva, Heidelberg, F.R.G. All chemicals were reagent grade. (c) Modification of pBR322 by N-AcO-AAIF Plasmid DNA (50 pg/100 pl) was modified in vitro by reacting it at 37°C with N-AcO-AAIF ( ~ 0"3 ~g/ml) in 2x 10 -5 M-citrate buffer (pH 7), containing 5~/o (v/v) ethanol. The modified DNA was extracted 3 times in chloroform and precipitated in ethanol saturated with sodium acetate as recommended by Fuchs et al. {1988). AAIF adducts in the DNA were quantified by ultraviolet light spectrophotometry on the basis of the AAIF absorbance peak at 310 nm, given the extinction coefficient of 23,000 (Lef~vre et al., 1978). The extents of modification in 2 DNA samples were estimated as 40"6 adducts/plasmid (A31o/A260 = 0"0167) and 104 adducts/plasmid (A3to/A26 o = 0"0429) after reaction times of about 15 rain and 45 min, respectively. (d) Mutation assay The spectrum of mutations induced by AAIF was studied in a forward mutation assay based on inactivation of the tetracycline resistance gene of pBR322 (Fuchs et al., 1981, 1983, 1988). This gene is 1188 base-pairs long and has BamHI and SalI restriction sites at positions 375 and 651, respectively. The 2 restriction sites therefore delimit a 276
A rapid boiling mini-prep procedure (Holmes & Quigley, 1981) was used to isolate plasmid DNA from 30 tetracycline-sensitive clones recovered from SOS-induced cells transformed with pBR322 containing about 6"5 AAIF adducts/6 S fragment (i.e. equivalent to about 100 adducts/plasmid). Plasmids that migrated faster than wild-type pBR322 in agarose-gel electrophoresis (1/30) or were not susceptible to digestion by BamHI and SalI (2/30) were eliminated from further analysis. Plasmid DNA for mutational analysis was isolated and purified by cesium chloride density gradient centrifugation. A I0 gg sample of the DNA was digested by BamHI and SalI, dephosphorylated with calf intestinal alkaline phosphatase, and end-labeled with [32P]ATP and T4 polynucleotide kinase. Single strands of the 6 S BamHI-SalI fragment were isolated by acrylamide gel electrophoresis, eluted from the gel, and precipitated in ethanol. The nueleotide sequences of the 6 S fragments were determined by Maxam & Gilbert chemistry (Maxam & Gilbert, 1977, 1980) followed by electrophoresis on 8% and 20% (w/v) acrylamide sequencing gels. Details of the materials and methods used in the forward mutation assay have been reviewed (Fuchs et al., 1988).
3. Results (a) Transformation and recovery of mutants
E. cell strain AB1157 was t r a n s f o r m e d with plasmids t h a t had been reconstructed b y ligating 6 S f r a g m e n t s containing 0, 2-5 or 6"5 A A I F a d d u c t s to the remainder of the plasmid f r o m unmodified pBR322. T r a n s f o r m a t i o n efficiencies were e s t i m a t e d from n u m b e r s of t r a n s f o r m a n t s per n a n o g r a m of t r a n s f o r m i n g D N A relative to control t r a n s f o r m a tions, m a k i n g the a s s u m p t i o n t h a t the efficiency of
G. R. Hoffmann and R. P. P. Fuche
242
Table 1
Induction of mutations by A A I F in the tetracycline-resistance gene of plasmid pBR322 AAIF adduets per 6 S
Bam-Sal
Plasmid
fragment
pBR322 control 16 S+6 S ligated 16 S+6 S ligated 16 S+6 S ligated 16 S+6 S ligated
SOS induction of host
Transforming efficiencyt
Relative transforming efficiency:~
Clones tested
Mutants
Mutant frequency (%)
_ + + + -
1"000 0'036 0.026 0"017 0"009
-1'00 0"72 0"47 --
31 315 865 1366 617
0 0 4 30 2
-0'00 0"46 2-20 0'32
0 0 2-5 6"5 6"5
t Relative to the pBR322 control. :~Relative to the unmodified ligated plasmid.
l i g a t i o n w a s t h e s a m e in t h e t h r e e s a m p l e s . T h e d a t a a r e p r e s e n t e d in T a b l e 1. T h e d e c r e a s e d t r a n s f o r m i n g efficiencies w i t h 2"5 or 6"5 A A I F a d d u c t s p e r p l a s m i d reflect t h e r e d u c t i o n o f v i a b i l i t y o f plasmids bearing these adducts. T o d e t e c t f o r w a r d m u t a t i o n s i n d u c e d b y A A I F in t h e t a r g e t 6 S BamHI-SalI r e s t r i c t i o n f r a g m e n t , i n d i v i d u a l clones w e r e t e s t e d for s e n s i t i v i t y t o t e t r a c y c l i n e (40 p g / m l ) . N o s p o n t a n e o u s t e t r a c y c l i n e s e n s i t i v e m u t a t i o n s w e r e d e t e c t e d in t h e s m a l l s a m p l e (315 clones) o f t r a n s f o r m a n t s f r o m u n m o d i fied p B R 3 2 2 t h a t w a s t e s t e d , b u t h i s t o r i a l c o n t r o l
d a t a ( F u c h s et al., 1988) i n d i c a t e t h a t t h e f r e q u e n c y o f s p o n t a n e o u s m u t a t i o n s in S O S - i n d u c e d cells is a b o u t 6 x l 0 -4. T h e d a t a in T a b l e 1 s h o w a dosed e p e n d e n t i n c r e a s e in m u t a t i o n f r e q u e n c i e s in p l a s m i d s m o d i f i e d w i t h A A I F t o t h e e x t e n t o f 2"5 a n d 6"5 a d d u c t s p e r 6 S f r a g m e n t . T h e p r o p o r t i o n s o f l e a k y m u t a n t s , d e f i n e d b y g r o w t h on t e t r a c y c l i n e a t 20 p g / m l b u t n o t a t 40 p g / m l , w e r e 1 o f 4 a t t h e l o w e r dose a n d 4 o f 30 a t t h e h i g h e r dose. T h e f r e q u e n c y o f m u t a n t s r e c o v e r e d a t t h e h i g h e r dose is a b o u t 35-fold t h e e x p e c t e d s p o n t a n e o u s f r e q u e n c y . The m u t a t i o n experiments were conducted with
Table 2
Mutations induced by A A I F in the B a m - S a l fragment of the tetracycline-resistance gene of plasmid pBR322 Position in the Tc' genet
Mutation number
Mutation:[: Description of the site of the mutation
390 400-401 402-403 419-420
5-53 13-103 11-87 16-28b
G--*C -G --C +G
420 428 439-440
5-111 6-82 16-28a
C--*G G--*T -CT
461 474 520-521
1-108~ 6-125 6-61
G--*T C--*A -G
528 530 532-533 536 537 536-540 536-540 536-540 537-540 549-552 573 573-574
6-63 1-100~ 5-59 14-112a 6-31 11-120 1-42 12-101 14-112b 1-67 4-113§ 7-26
C-~G G-~C - G G-*T G-*T +G -G -G -G -GC T --*C +C
586-587
4-76
-G
613-615
1-56
-G
GG dinucleotide in the sequence CCG_GAC GG dinucleotide in the sequence GTG_GGCC CC dinueleotide in the sequence GGCCGG G insert between repetitive CA dinucleotides in the sequence CACA In repetitive CA dinucleotides of the sequence CACA GG dinucleotide in the sequence CGGTT Loss of dinucleotide from sequence C-C__T_TAadjacent to a NarI site Run of 4 G residues Adjacent to run of 3 G residues GG dinucleotide in the sequence TGGTGG; a hotspot for mutagenesis by AAF Run of 4 C residues Adjacent to run of 4 C residues GG dinucleotide in the sequence TG__GC Run of 5 G residues Run of 5 G residues Run of 5 G residues Run of 5 G residues Run of 5 G residues Run of 5 G residues NarI site (GGCGCC); a hotspot of mutagenesis by AAF TT dinucleotide in the sequence ATTCC C insert between TT and CC dinucleotides in the sequence TTCC GG dinucleotide adjacent to a run of trinucleotides: GCGGCGGCGG Run of 3 G residues
t Numbering as defined by Sutcliffe (1979). :~All mutations are described for the DNA strand whose sequence corresponds to that of mRNA. § Phenotypieally leaky (grows at 20 #g tetracycline/ml but not at 40 pg tetracyeline/ml).
Coding change Gly~Ala - 1 frameshift - 1 frameshift + 1 frameshift Thr--*Arg Val-*Phe - 2 frameshift Gly-*Trp Ala-~Asp - I frameshift Pro--*Arg Val-*Leu - 1 frameshift Gly--*Trp Gly-*Val + l frameshift - 1 frameshift - 1 framesbift - 1 frameshift - 2 frameshift Phe --*Ser + 1 frameshift - 1 frameshift - 1 frameshfft
AAIF
Mutation Spectrum
243
Table 3
Comparison of the mutation spectrum of A A I F to the spectra of A A F and A F AAIF
AAF~f
AF:~
A. Classification of the mutations (l) Frameshift mutations + 1 frameshifts 1 frameshifts 2 frameshifts 3 deletions~ (2) Base-pair substitutions Transitions Transversions GC to TA GC to CG AT to TA
14/24
32/34 3/14 9/14 2/14 0/14
-
-
-
10/24
6/23 2/32 13/32 12/32 5/32
2/34 1/ 10 9] 10 5/9 4]9 0/9
0/6 4/6 2/6 0/6 17/23
1/2 1/2 0/1 0/1 1/1
1/ 17 16/17 14/16 1/16 1/16
B. Sites of the mutations (1) Mutations initiated at a G-C base-pair (2) Mutations initiated at an A ' T base-pair
23/24 1/24
33/34 1/34
22/23 1/23
0/24 1/24 23/24
0/34 12/34 22/34
4/23 2/23 17/23
C. Sequences surroundinq the sites of the mutations (1) Mutations in non-repetitive, sites (2) - 2 frameshffts in NarI'sites (GGCGCC) (3) Mutations associated with a repetitive site (i) Extent of repetition: Run of 5 bases Run of 4 bases Run of 3 bases Run of 2 bases Repetitive dinucleotides (e.g. CACA) Repetitive trinucleotide (--3 in GCGGCGGCG) (ii) Association with the repetition: Frameshifts at repetitive sites Proportion of frameshffts at repetitive sites Proportion of frameshifts in runs of 3, 4 or 5 bp Deletion of repetitive trinucleotide Base-pair substitutions at repetitive sites Proportion of substitutions at repetitive sites Proportion of substitutions in runs of 3, 4 or 5 bp Mutations immediately adjacent to a run of 3, 4 or 5 bp Insertion, deletion or substitution between two repeats (e.g. TTXCC)
6/23 3/23 2/23 10/23 2/23 0/23 12/23
2/21 4/21 1/21 9/21 1/21 5/21 15/21
12/14 5/12 0/23 8/23
1/17 1/17 7/17 8/17 0/17 0/17 3/17
15/32 7/15 5/21 2/21
8/10 4/8
3/6 1/3 0/17 11/17
2/2 0/1
11/17 7/11
2/23 1/23
0/21 0/21
1/17 2/17
20/22 2/22
34/36 2/36
18/20 2]20
D. Occurrenceof complex or multiple mutationsll (1) Simple mutations (2) Complex or multiple mutations
J" Based on Koffel-Schwartz et al. (1984); includes data from wild-type and uvrA strains of E. coll. bp, base-pairs. J: Based on Bichara & Fuchs (1985). § Though not frameshifts in a strict sense, --3 mutations are pooled with frameshifts because they involve deletions of base-pairs (bp). ]1The multiple (or complex) mutations induced by AAIF and AF are included in the Table; the total numbers of mutations are therefore 24 and 23, respectively. The complex mutations induced by AAF are omitted because they are not readily described as a combination of simple mutations.
cells w h o s e S O S s y s t e m h a d b e e n i n d u c e d b y u l t r a v i o l e t l i g h t , b e c a u s e m u t a g e n e s i s b y A A F (KoffelS c h w a r t z et al., 1984) a n d A F ( B i c h a r a & F u c h s , 1985) is k n o w n t o be d e p e n d e n t on S O S f u n c t i o n s o f t h e host. T r a n s f o r m a t i o n i n t o u n i n d u c e d cells revealed that mutagenesis by AAIF similarly d e p e n d s on h o s t S O S f u n c t i o n s . T h e d e p e n d e n c e , m o r e o v e r , m a y b e g r e a t e r t h a n t h e s e v e n f o l d difference s h o w n in T a b l e 1, b e c a u s e t h e p u t a t i v e m u t a n t s r e c o v e r e d in t h e S O S - i n d u c e d cells w e r e confirmed by subsequent analysis, while the two m u t a n t s r e c o v e r e d in t h e u n i n d u c e d cells w e r e n o t analyzed further.
(b) Sequence analysis of mutations induced by A A I F S e q u e n c e a n a l y s i s w a s p e r f o r m e d o n 22 m u t a n t s from plasmids that had been modified with AAIF to t h e e x t e n t o f 6"5 a d d u c t s p e r B a m t t I - S a l I f r a g m e n t . T h e m u t a t i o n s a r e l i s t e d in T a b l e 2. T h e T a b l e s h o w s 24 m u t a t i o n s b e c a u s e p l a s m i d s 1 4 - 1 1 2 a n d 16-28 turned out to be double mutants. The fact t h a t all t h e m u t a t i o n s e x c e p t o n e a r e i n i t i a t e d a t a G-C base-pair suggests that the mutations are t a r g e t e d a t s i t e s o f A A I F a d d u c t s on g u a n i n e residues. Table 2 reveals that the majority of AAIF-
244
G. R. Hoffmann and R. P. P. Fuchs
induced mutations are transversions and frameshift mutations involving the loss of a single base-pair. Frameshift mutations involving the loss of two base-pairs or the gain of a single base-pair were also observed. The data also reveal that the mutations are strongly associated with repetitive sequences, including a prominent hotspot at position 536 to 540, which is a run of five G'C base-pairs. The classes of mutations that comprise the mutation spectrum of AAIF are summarized and compared to the mutation spectra of AAF and AF in Table 3. The most conspicuous difference among the mutation spectra is the relative proportion of frameshift mutations and base-pair substitutions. The ratio of substitutions to frameshifts was l0 : 14 forAAIF, 2 : 3 2 for AAF (Koffel-Schwartz et al., 1984), and 17 : 6 for AF (Bichara & Fuchs, 1985). Contingency X2 calculations show the distribution of substitutions and frameshifts induced by AAIF to differ significantly from that induced by either AAF or AF. 4. Discussion
The frequencies of mutations induced by AAIF in SOS-induced cells (Table 1) correspond to 0"0018 mutation per adduct in plasmids carrying 2"5 adducts per 6 S fragment and 0"0034 mutation per adduct in plasmids with 6"5 adducts per 6 S fragment. The difference between the two dosages suggests that the efficiency with which AAIF adducts are processed into mutations is higher when more adducts are present. These mutagenic efficiencies are similar to those reported for AAF, where there were 0"0019 and 0"0025 mutation per adduct at 3 and 6 adducts per 6 S fragment, respectively (Koffel-Schwartz et al., 1984). Although AAIF adducts are comparable to AAF adducts in mutagenic potency, they differ from AAF adducts with regard to the kinds of mutations induced. Whereas AAF induces frameshift mutations almost exclusively (Koffel-Schwartz et al., 1984), AAIF induces frameshift mutations and base-pair substitutions in roughly equal numbers. It is likely that the proportion of base-pair substitutions shown in Table 3, like that in any assay that detects mutations on the basis of phenotype, is underestimated because some base-pair substitutions may be silent or leaky enough to preclude detection. Nevertheless, the observation that about 50o/o of AF-induced mutations are phenotypically leaky (Bichara & Fuchs, 1985), but only about 15% of AAIF-induced mutations are leaky by the same criterion, is consistent with the sequencing data (Bichara & Fuchs, 1985) in indicating that AF is more specific than AAIF as a base-pair substitution mutagen. The comparative data therefore place the mutation spectrum of AAIF intermediate between those of AAF and AF. Rather than simply being intermediate, the mutation spectrum of AAIF has properties that differ from both AAF and AF. Conclusions in this respect are somewhat tenuous because of limited
numbers of mutations sequenced, and additional differences or spectral complexities might be revealed if the analysis for all three compounds were enlarged. Nevertheless, certain trends are evident from a comparison of the mutation spectra summarized in Table 3. Besides containing more frameshift mutations, the mutation spectrum of AAIF differs from that of AF in the proportion of different classes of base-pair substitutions. Both agents induce transitions only rarely; however, the spectrum of AAIF includes G" C to T ' A transversions and G'C to C'G transversions in equal numbers, while that of AF includes G" C to T" A transversions almost exclusively. Similarly, the distribution of frameshift mutations induced by AAIF differs from that induced by AAF. The frameshift mutations induced by AAIF are principally - 1 (9/14) and + 1 (3/14) frameshifts, and they are strongly associated with repetitive guanine residues. Repetitive sequences are also hotspots for mutagenesis by AAF (Koffel-Schwartz et al., 1984); however, the spectrum of frameshift mutations induced by AAF includes a high frequency of - 2 frameshifts in N a r I sites (GGCGCC) (Koffel-Schwartz et al., 1984). Only one such mutation was found among 24 AAIF-induced mutations. Multiple mutations in the tetracycline resistance gene seem to occur at a frequency higher than that expected for independent events. In the current study, mutant 16-28 contains a + 1 frameshift at position 419 to 420 and a - 2 frameshift at position 439 to 440. Mutant 14-112 has a G ' C to T ' A transversion at position 536 and a - 1 frameshift at position 537 to 540. In view of the proximity of the sites, it seems likely that mutant 14-112 represents a single complex mutation in which the two central guanine residues in the sequence CCGGGG were replaced by a thymine. Complex mutations and multiple mutations have been observed repeatedly in the forward mutation assay in the tetracycline resistance gene: two induced by AAIF, two by AAF (Koffel-Schwartz et al., 1984), two by AF (Bichara & Fuchs, 1985) and four by cisplatin (Burnouf et al., 1987); in each case, the sample sizes and mutation frequencies would support a prediction of about 0"5 multiple mutation under the assumption that the mutations are independent events. The mechanisms underlying the complex and multiple mutations are obscure. Some of the differences in mutation spectra of AAF, AF and AAIF can be interpreted with respect to conformational changes that the compounds induce in DNA. AAF adducts cause a major conformational change, described by an insertiondenaturation model (Daune et al., 1981). In contrast, AF causes a less severe conformational change that may be ascribed to insertion without denaturation or outside binding (Bichara & Fuchs, 1985; Daune et al., 1981). Analyses of DNA structure (Fuchs & Daune, 1973; Fuchs et al., 1976; Lang et al., 1979; Lef6vre et al., 1978) suggest that AAIF does not cause the severe distortion associated with AAF, presumably because the iodo group sterically
AAIF
245
Mutation Spectrum
interferes with insertion. As might be expected from the structural data, the analysis of mutations induced by AAIF reveals a mutation spectrum that resembles that of AAF in some respects and AF in others. Several lines of evidence suggest that the - 2 frameshift mutations induced by AAF in NarI sites (G1G2CG3CC) are associated with destabilization of the B structure of DNA in the vicinity of AAF adducts on the third guanine residue, forming a local Z-like structure (Koffel-Schwartz et al., 1984; Burnouf et al., 1989; Koehl et al., 1989). In contrast to AAF adducts, AAIF adducts are less effective in inducing - 2 frameshift mutations in N a r I sites. The explanation for this difference between the two mutagens may lie in the differences in the conformational changes that they cause. The AAF adduct causes the guanine to rotate from the anti to the syn conformation (Daune et al., 1981; Evans et al., 1980). This conformational change may trigger the B to Z transition in stretches of alternating GC residues; elsewhere, it may cause the local denaturation associated with insertion of the fluorene ring and extrusion of the guanine. In contrast, the rotation of guanine from anti to syn seems to be sterically blocked in the case of AAIF adducts (Lef~vre et al., 1979). Consequently, AAIF adducts should be less effective than AAF adducts in stimulating the B to Z transition that appears to be centrally involved in the N a r I mutation pathway. The degree to which repetitive sequences are hotspots seems to depend on the length of the repeat; this observation is consistent with mutation spectra obtained in other systems (Ripley et al., 1986). The longest repeat in the target fragment used in our study is the run of five guanine residues at site 536 to 540. This single run accounts for one-fourth of the mutations induced by AAIF. Correcting for the number of occurrences of runs of different lengths, one observes t h a t the run of five guanine residues is more mutable than runs of four, and runs of four guanine residues are more mutable than those of two or three. Eveh runs of two or three guanine residues, however, are more mutable than isolated guanine residues. The slippage model (Streisinger & Owen, 1985; Strei§inger et al., 1966) can explain the high susceptibility of repetitive sequences to frameshift mutagenesis by AAIF. The data in Table 2, however, suggest that repetitive sequences are hotspots not only for frameshift mutations but also for ,transversions induced by AAIF. A tendency for transversions to be more frequent in runs than in nonrepetitive sequences can also be discerned among the mutations induced by AF in the same assay (Bichara & Fuchs, 1985; Table 3). Recent evidence suggests that a template dislocation model c a n explain the occurrence of some transversions at sites of repetitive bases (Boosalis et al., 1989; Kunkel & Alexander, 1986). According to this model, a transient misalignment during replication leads to the misincorporation of a base at the site of the 5'-most base in the template strand of a short run; the new
base is complementary to the next base in the template. Although the model has been considered principally for hotspots of spontaneous mutagenesis, it could also be applicable to induced mutations. Inspection of the sequences surrounding the transversions induced by AAIF adducts reveals that only mutation 1-108 clearly conforms to the model. One may speculate therefore t h a t other factors must contribute to the tendency for transversions to occur preferentially at sites of repetitive bases. In this respect, it has recently been shown (Seeberg & Fuchs, 1990) that AAF adducts on the three guanine residues in the sequence 5'-GtG2CGaCC-3' are not equally susceptible to incision by the u v r A B C excinuclease. Although the result may be specific to this particular sequence, the reduced incision when AAF adducts are on G2 suggests the possibility that adducts on guanine residues may be less efficiently repaired in general when the adjoining 5' nucleotide is also a guanine. I f so, it is conceivable that the difference in reparability of adducts in runs of guanine residues or in GG dinucleotides may also contribute to these sequences being hotspots for mutagenesis by AAIF. We thank Professor Michel Daune whose insight into the structural effects of AAIF provided the impetus for studying its mutagenicity. We also thank Marc Bichara, R~gine Bmtz, Dominique Burnouf, Anne-Marm Freund, Patrice Koehl and Xavier Veaute for valuable discussions and collaboration during the course of this study and Mme Liliane Diebolt for excellent secretarial work on the manuscript. This work was supported partly by grants from the Association pour la Recherche sur le Cancer (no. 6143), from the F~d~ration Nationale des Centres de Lutte contre le Cancer and from the MRES "action sondes froides." The support of a fellowship from the NIH French CNRS Program for Scientific Collaboration and sabbatical support from the College of the Holy Cross, Worcester, MA, U.S.A., are gratefully acknowledged• •
e
,
.
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Edited by P. Chambon