Chem. Res. Toxicol. 1993,6, 819-824
819
Nucleotide Misincorporation on DNA Templates Containing N-(Deoxyguanosin-NL-yl)-2-(acety1amino)fluorene Shinya Shibutani’ and Arthur P. Grollman Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651 Received June 23,1993”
An octadecadeoxynucleotide, modified site-specifically with N-(deoxyguanosin-P-y1)-2(acety1amino)fluorene (dG-N2-AAF),was prepared by enzymatic synthesis from a comparably modified decamer and then used as a DNA template in primer extension reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I containing (exo+) or lacking (exo-) 3/45’exonuclease activity. Using exo- Klenow fragment and all four deoxynucleotide triphosphate (dNTPs), primer extension is blocked one base before and opposite dG-N2-AAF. A small fraction of the reaction product represents translesional synthesis, in which dAMP is incorporated opposite the lesion. Kinetic studies of base insertion and chain extension indicate that the frequency of dAMP insertion opposite dG-N2-AAF is higher than that of other deoxynucleotide monophosphates (dNMPs) and of N-(deoxyguanosin-8-y1)-2-(acetylamino)fluorene (dG-C8-AAF); however, the rate of extension of dA.dG-N2-AAF from the 3’ terminus was much lower than that of dA.dG-C8-AAF. We conclude that dG-N2-AAF is a miscoding lesion and capable of generating G 4 T transversion mutations in cells.
Introduction Aromatic amines are recognized us environmental pollutants; some have been shown to be carcinogenic in experimental animals and humans (1,2). (Acety1amino)fluorene is widely used as a prototype aromatic amine in studies of chemical carcinogenesis (3). Metabolicallyactivated forms of (acety1amino)fluorene react with DNA to produce a variety of covalent adducts, including those shown in Figure 1. The major adduct formed in vitro is N-(deoxyguanosin-8-yl)-2-(acetylamino)fluorene (dG-CBAAF) ( 4 ) . N-(Deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) is predominantly found in vivo ( 5 , 6 ) . Five to fifteen percent of DNA-bound aminofluorene in vitro and in vivo is recovered as N-(deoxyguanosin-Wyl)-2-(acetylamino)fluorene (dG-N2-AAF)(6-1 1). dG-N2AAF has been shown to persist in the tissues of animals treated with this carcinogen while dG-C&AAF and dGC8-AF are readily excised by DNA repair enzymes (6, 7, 4,8, 12). The mutagenic potential of dG-C8-AAF and dG-C8-AF has been explored in bacteria and mammalian cells using oligodeoxynucleotides containing site-specific modifications (13-1 9). These modified oligodeoxynucleotides also have been used as templates for primer extension experiments in vitro (20, 21). dG-N2-AAFhas been difficult to prepare in the form of a site-specifically modified oligodeoxynucleotide, and the mutagenic potential and spectra of this adduct have previously not been reported. We have isolated by HPLC a decadeoxynucleotide containing a single dG-N2-AAF ~~~
~
~
~~
~
* To whom correspondence should be addressed. Telephone:
444-3080; Telefax: (516)444-7641.
(516)
0 Abstract published in Advance ACS Abstracts, October 1, 1993. 1 Abbreviations: AAF, 2-(acetylamino)fluorene;dG-N2-AAF,N-(deoxyguanoain-W-yl)-2-(acetylamino)fluorene; dG-C8-AAF, N-(deoxyguanosin-&yl)-2-(acetylamino)fluorene;dG-C&AF,N-(deoxyguanosin-8-y1)2-aminofluorene;N-acetoxy-AAF,N-acetoxy-2-(acetylamino)fluorene;dG, 2’-deoxyguanosine;pol I, DNA polymerase I; exo-, 3’ 5’exonucleasefree Klenow fragment; exo+, intact Klenow fragment; Fh,frequency of base insertion; F, frequency of chain extension; dNTP, 2’-deoxynucleotide triphosphate, PAGE, polyacrylamide gel electrophoresis.
-
-
dG N2 - A A F
dG-CB- A A F
Figure 1. Structures of dG-N2-AAFand dG-C8-AAF.
(22). Extending the sequence by enzymatic synthesis, this modified DNA template was used to determine kinetic parameters by which deoxynucleotide triphosphate (dNTPs) are incorporated opposite dG-N2-AAFand the rate of extension from the 3’ terminus of base pairs containing the lesion. These studies provide insight into the miscoding properties and mutagenic potential of dGN2-AAF.
Experimental Procedures Materials. Organic chemicals used for the synthesis of oligodeoxynucleotideswere supplied by Aldrich Chemical (Milwaukee, WI). [yS2P]ATP(specificactivity >5000 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL). Cloned exo- (specificactivity 21 200 units/mg of protein) and exo+(17 400 unita/mg) Klenow fragment of Escherichia coli DNA polymerase I (pol I) were purchased from United States Biochemical Corp. (Cleveland,OH). T4 polynucleotide kinase was from Stratagene (La Jolla, CA), and 2’-deoxynucleotide triphosphates were from Pharmacia (Piscataway, NJ). A Waters 990 HPLC instrument, equipped with a photodiode array detector, was used for separation and purification of modified and unmodified oligodeoxynucleotides. Synthesis and Purification of Oligodeoxynucleotides. Unmodified oligodeoxynucleotideawere prepared using a Dupont Coder 300 automated DNA synthesizer (23) and purified on a reverse-phase column, WBondapak CIS (0.39 X 30 cm, Waters), eluted over 60 min at a flow rate of 1.0 mL/min with a linear gradient of 0.05 M triethylamine acetate (pH 7.0) containing 10-15 % acetonitrile (22). Oligodeoxynucleotideswere further purified on 20% polyacrylamide gel (15 X 72 X 0.2 cm) in the presence of 7 M urea. Bands located under ultraviolet light were
0893-228~/93/2706-0819$04.00/0 0 1993 American Chemical Society
820 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
Table I. Sequence of Oligodeoxynucleotides. 5'+3' sequence CACTAGTCACTTTCCTCT CACTAG*TCAC CACTAG*TCACT?TCCTCT AGAGGAAAGTGANTAGTG AGAGGAAAGTGATAGTG AGAGGAAAGT AGAGGAAAGTGA AGAGGAAAGTGAN
no.
Shibutani a n d Grollman
Y+dCTP
32P-CACTA X TCAC GTGAT N AGTGAAAGGAGAS'
x. -
N~-AAF N~-AAF
c
N
n
C~-AAF
c -
A
-
0.1 0.01 0.1 0.01
0.1
0.01
7
8
u
0 Sequence of templates and primers. G* = dG-N2-AAF,dG-C8 AAF, or dG-C8-AF; N = C, A, G, or T.
extracted with 2.0 mL of distilled water overnight a t 4 "C and then concentrated on a Centricon 3 filter (Amicon) by centrifugation a t 5000 rpm. Samples were subjected to HPLC, as described above, to remove urea. Oligodeoxynucleotides were labeled a t the 5' terminus by treating with bacteriophage T4 polynucleotide kinase in the presence of [ T - ~ ~ P I A T(24). P The product was applied to 20% polyacrylamide gel (35 X 42 X 0.04 cm) in the presence of 7 M urea to establish homogeneity. Positions of oligomerswere established by autoradiography, using Kodak X-Omat XAR film. Sequences of oligodeoxynucleotides used in these experiments are listed in Table I. Preparation of a DNA Template Containing dG-N2-AAF and dG-CS-AAF. Decadeoxynucleotidescontaining a single dGN2-AAFor dG-C8-AAF (sequence 2) were prepared as described previously (22). 32P-Labeleddecamers, modified with dG-N2AAF or dG-C8AAF (0.2 pM), were annealed to an 18-mer (sequence4,0.5 pM) containing dC or dA opposite the lesion and incubated for 1.5 h a t 10 "C in a solution containing dCTP and dTTP (100 pM each), 50 mM Tris-HC1 (pH 8.0), 8 mM MgCl2, 2-mercaptoethanol(5 mM), and 0.01 or 0.1 unit of exo- Klenow fragment. A scaled-up reaction was incubated for 1.5 h a t 10 "C in reaction mixtures (1.5 mL) containing unlabeled dG-N2-AAF'modified 10-mer primer (1pg), annealed to an 18-mer template containing dA opposite the adducted base (4.5 pg), dCTP and dTTP (100 pM each), and 15 units of exo- Klenow fragment. Samples were heated for 3 min a t 95 "C and concentrated on a Centricon 3 filter. The dG-N2-AAF-modified18-mer (sequence 3) was separated by HPLC, as described above. A dG-C8-AAFmodified 18-mer was prepared by reacting an unmodified 18mer (sequence 1)with N-acetoxy-AAF (22). Primer Extension Studies. Primer extension reactions were conducted for 1h at 25°C in mixtures containing four dNTPs (100 pM each), 0.1 pM unmodified or dG-N2-AAF-modified templates primed with 0.05 pM 32P-labeled10-mer,and varying amounts of DNA polymerase. Samples were heated for 3 min a t 95 "C in the presence of formamide dye and then subjected to 20% polyacrylamide gel (15 X 72 X 0.04 cm) in the presence of 7 M urea. Bands were identified by autoradiography. Radioactivity was measured in a Packard scintillation counter using Liquiscint (National Diagnostics,Manville, NJ). Standards (sequences 4 and 5) were partially digested a t 25°C for 2 min, using 1.0 X 106units of venom phosphodiesterase I (Sigma, St. Louis, MO) in 10 p L of 100 mM Tris-HC1 buffer (pH 8.0)to establish the relative migration of incompletelyextended primers. Base Insertion and Chain Extension. Kinetic parameters of base insertion opposite the lesion and chain extension from the 3' terminus were determined under conditions similar to those described for the primer extension assay (25). Reaction mixtures containing varying amounts (0.0005-0.5 unit) of exo- Klenow fragment were incubated at 25°C for 1-6 min in reactions containing 10 pL Tris-HC1 (pH 8.0) and 0.1 pM DNA template (5'-CACTAXTCACTTTCCTCT; X = dG or dG-N2-AAF)primed with 0.05 pM 3?P-labeled12-mer(sequence7)for insertion kinetics and a 13-mer (sequence 8) for chain extension kinetics. Nucleotide insertion kinetics were measured in reactions catalyzed by exo- Klenow fragment using 0.0005 unit for 90 s (COGpair), 0.01 unit for 90 s (TOG), 0.025 unit for 90 s (GoG), 0.025 unit for 6 min (A-G), and 0.05 unit for 6 min (N*dG-N2-AAFpairs). Kinetics of extension were measured using the following amounts of
18-
101
2
3
4
5
6
Figure 2. Enzymatic extension of dG-N2-AAF-or dG-C8-AAFmodified decamer. 32P-Labeled10-mer containing dG-N2-AAF or dG-C8-AAF (lanes 1and 6) primed with 18-mer (sequence 4), containing dC (lanes 2,3,7, and 8) or dA (lanes 4 and 5) opposite the lesion, was incubated for 1.5 h a t 10°C in the presence of dCTP, dTTP, and 0.01 or 0.1 unit of exo- Klenow fragment, as described under Experimental Procedures. enzyme: 0.0005 unit for 90 s (COG),0.005 unit for 90s (TOG),0.05 unit for 6 min (A-G, GoG), and 0.5 unit for 6 min (N*dG-N2-AAF pairs). All reaction rates were linear for the 6-min reaction period used in our experiments. Samples were heated a t 95°C for 3 min in the presence of formamide and then subjected to 20% polyacrylamide gel electrophoresis (PAGE) (35 X 42 X 0.04 cm) in the presence of 7 M urea as described above. The Michaelis constant (Km) and maximum rate of the reaction (V-) were obtained from Hanes-Woolf plots of the kinetic data. Insertion (Fh)and extension (Pea)frequencies were determined relative to dC-dGaccording to equations developed by Mendelman et al. (26,27) and our previous report (25),where F = ( V,JK,) [wrong pair]/( VmJKm) [right pair], with "wrong pair" defined as a base mismatch or any base pair containing dG-N2-AAF. All reaction rates were linear over the course of the experiment; data reported represent the average of 2-4 separate experiments in which less than 20% of the primer is extended (28).
Results Preparationof Template OctadecamerContaining dG-N2-AAF.A decadeoxynucleotidecontaining a single dG-N2-AAFor dG-C8-AAF was extended on a primed 18mer to form a dG-N2-AAF- or dG-C8-AAF-modified 18mer, as shown in Figure 2. When an 18-mer containing dC opposite dG-N2-AAFwas used (lane 2), the amount of dG-N2-AAF-modified18-mer formed represented 16% of the starting primer. Using an 18-mer containing dA opposite the lesion (lane 4), the amount of dG-N2-AAF modified 18-mer increased to 85% of the starting primer. When excess enzyme or longer incubation time was used, blunt-end extension of the fully extended product containing dG-N2-AAF was observed. The dG-C8-AAF modified 18-mer (lane 7) was produced efficiently, using small amounts of Klenow fragment (lane 8). In purifying
Chem. Res. Toxicol., Vol. 6, No. 6,1993 821
Nucleotide Misincorporation by d G - P - A A F
dNTPs
.03-
TG%ACTAXTCAC c
Unmod.
.02-
N~-AAF exo+
0 u) N
A
Y
N2 -AAF- 18mer
W 0
z
2c .01-
N2-AAF-10mer
-32P
z-
A G
1 8 ~18c 1& 181 17a
I 18-
0
cn
m
a
17
-
16-
O r
15I
I
I
I
I
I
14Figure 3. HPLC separation of octadecamer containing dG-N2AAF. The HPLC system is described under Experimental Procedures.
1312-
Unmod.
N*-AAF C'-AAF
C*-AF
10-
I
1 2 3 4 Figure 4. Polyacrylamide gel electrophoresis of octadecamers containing a single AAF-derived adduct. A dG-N2-AAF-18-mer (lane 2) and dG-C8-AAF-18-mer (lane 3) were prepared byprimerextension reactions using their modified 10-mer.Unmodified 18mer (lane 1)and dG-C8-AF-18-mer (lane 4) were prepared by chemical synthesis. Oligodeoxynucleotideswere labeled with S 2 P as described under Experimental Procedures and then subjected to electrophoresis on 20% polyacrylamide gel (15 X 72 X 0.04 cm) for 24 h at 1500 V.
the products of the scaled-up reaction (Figure 3), a dGN2-AAF'-modified 18-mer (sequence 3, t R = 48.6 min) was separated by HPLC from its complementary strand ( t R = 18.5 min) and from the dG-N2-AAF-modified 10-mer starting material ( t R = 40.9 min). Incompletely-extended products of dG-N2-AAF-modified 10-mer eluted at a position between the dG-N2-AAF-modified 10-mer and 18-mer. The UV spectrum of dG-N2-AAF-modified 18-mer (data not shown) was similar to that of the dG-N2-AAF-modified 10-mer reported previously (22). Homogeneity of the dGN2-AAF' modified 18-mer was established by electrophoresis on 20 % polyacrylamide gel containing 7 M urea, following 5'-labeling of the oligodeoxynucleotide with 32P, as shown in Figure 4. The dG-N2-AAF-18-mermigrated more slowly than the unmodified 18-mer and faster than either the dG-CS-AAF-18-mer or the dG-C8-AF-18-mer. Migration of the dG-C8-AAF-18-mer (lane 3)was identical to that of the dG-C8-AAF 18-mer prepared postsynthetically (data not shown) (22). The dG-N2-AAFmonomer
1 2 3 4 5 6 7 8 9 1 0 1 1 Figure 5. Primer extension in the presence of four dNTPs. Reaction mixtures contained an unmodified 18-mer (sequence 1,O.l p M ) or dG-N2-AAF-modified18-mer (sequence 3,O.l p M ) as template, primed with 32P-labeled10-mer .(sequence 6, 0.05 pM), dNTPs (100 pM each), and either exo+ or exo- Klenow fragment (1 unit), in a final volume of 10 pL. Reactions were incubated for 1 h a t 25 "C, as described under Experimental Procedures. MA, 18C, 18G, 18T,and 17Aare synthetic standards containing the base (sequence 4) or one-base deletion (sequence 5), partially digested at 25°C for 2 min by venom phosphodiesterase I.
and its modified 18-mer were stable in solution under neutral conditions (pH 6.0-9.0). Primer Extension Studies.As shown in Figure 5,DNA synthesis on unmodified templates, catalyzed by the exo(lane 2) or exo+ (data not shown) Klenow fragment, as tested in the presence of four dNTPs, led to the expected incorporation of dCMP opposite the unmodified dG residue at position 13. Using a dG-N2-AAF-modified template and exo- Klenow fragment (lane 5), primer extension was blocked one base before (position 12) and opposite dG-N2-AAF (position 13). Maxam-Gilbert sequence analysis (29) of the blocked primers at position 13 confirmed incorporation of dAMP in the lower band and dGMP in the upper band (data not shown). A small amount (0.1 % of the starting primer) of the fully-extended 18-merwas detected in reactions containing all four dNTPs (lane 5); 0.2% (lane 6) of the fully-extended product was detected when dCTP was omitted. Sequenceanalysis (29) confirmed that dAMP was incorporated opposite dG-N2AAF lesion (Figure 6). When the exo+ Klenow fragment was used, primer extension was strongly blocked one base before dG-N2-AAF(Figure 5,lane 3). Deletions were not detected. Frequency of Nucleotide Insertionand Extension. The kinetic parameters of dNMP insertion and chain extension from the 3' terminus, catalyzed by exo- Klenow
822 Chem. Res. Toxicol., Vol. 6, No. 6, 1993
Shibutani and Grollman
Table 11. Kinetic Parameters of Nucleotide Insertion and Chain Extension Reactions Catalyzed by Klenow Fragment. base insertion: dNTP yA-=P S-CACTAXT-
chain extension: CrrrP y NA-=P 5'CACTAXT-
N*X Km (NM) V,, (% min-1) Fill9 K m (NM) V,, (7% min-1) Fat COG 3.38 i 0.03 40.2 f 3.8 1.0 3.76 f 0.76 23.3 f 1.65 1.0 A*G 59.7 i 1.6 0.41 f 0.01 5.74 X lo-' 16.1 f 3.82 (1.64 f 0.15) X le2 1.64 X lo-' G*G 50.1 f 3.5 4.22 i 0.13 7.08 X 10-3 70.4 f 12.9 0.21 i 0.03 4.82 X 1P TOG 33.4 f 10.8 0.21 f 0.12 5.16 X lo-' 55.8 f 1.63 1.63 f 0.08 4.71 X 10-3 27.6 f 8.6 (1.35 f 0.11) X le2 4.29 X 106 C*dG-N2-AAF ND ND A*dG-N2-AAF 14.1 f 2.3 0.18 f 0.06 1.06 X 103 53.8 i 10.3 (1.90 f 0.09) X lo-' 5.70 X le7 G*dG-N'-AAF 35.7 f 11.2 (1.14 f 0.09) X le2 2.78 X 106 ND ND T*dG-N2-AAF 73.2 f 2.2 (6.87 f 0.50) X 10-3 7.88 X 1o-S ND ND a Kinetics of insertion and extension were determined in reactions catalyzed by exo- Klenow fragment as described under Experimental Procedures. F = (V A K , ) [wrong pair]/( V&Km) [right pair = dC-dG1. ND = not detectable.
G
A + G
Template
G (T)
G A
A G (T)
G A
5' C A C T
T C A C
T
A
T
A 5'
T
Figure 6. Sequence analysis of fully-extended primer on dGN2-AAFmodified template. Fully-extended products recovered from 20 reactions, incubated under conditions described in the legend to Figure 5, were combined, and the sequence of the extended product was analyzed as described by Maxam and Gilbert (29).
fragment, were measured during translesional synthesis (Table 11). K m (14.1) and V m u (0.18) values for dAMP insertion opposite dG-N2-AAFwere 2-5 times lower and 13-26 times higher than for other dNTPs, respectively. The frequency of insertion (Fim)for dATP opposite dGN2-AAF (1.06 X 103)is 25-135 times higher than that for other dNTPs and 2 times higher than Fi, for dATP opposite unmodified dG. In the chain extension reaction, Fext could be detected only for the dA*dG-N2-AAFpair (5.70 X W7).
Discussion At least six different DNA adducts have been detected in animals and cells treated with 2-AAF; these include acetylated and deacetylated dG-C8 adducts (2), several oxidation products (30), and the dG-N2-AAFadduct (7). The major aminofluorene adducts formed in vivo are dGC8-AF and dG-C8-AAF; dG-N2-AAFrepresents 5-15 % of the total adducted DNA (6-8). The relative amounts of oxidation products formed are low (31,32); the degree to which these adducts are generated during isolation procedures has not been established. To relate the structure of damaged DNA to observed mutagenic events, experimental systems have been developed to help define the mutagenic potential of a single
lesion in vitro(33). dG-C8-AF and dG-C8-AAF have been introduced, site-specifically, into shuttle plasmid vectors and used to transform or transfect cells in culture (13-19); these adducts were also used for in vitro primer-extension studies catalyzed by DNA polymerases (20,21). dG-C8AAF is believed to generate base substitutions and deletions with the relative distribution of these events, depending on nucleotide sequence context, the nature of the vector, and proteins induced during the SOS response in bacteria. Site-specific studies of dG-N2-AAFhave not been reported, due to the lack of an efficient method for preparing oligodeoxynucleotides containing this adduct. By combining postsynthetic chemical modification procedures with enzymatic synthesis, we obtained sufficient material to demonstrate miscoding during DNA synthesis on DNA templates modified by dG-N2-AAF(22). The base incorporated opposite dG-C8-AAF was determined by primer-extension reactions catalyzed by the Klenow fragment of DNA polymerase I. Chain extension was blocked opposite the lesion; limited translesional synthesis was observed (Figure 5). During PAGE, the full length reaction product migrated as an oligodeoxynucleotide containing dA opposite the lesion (Figure 5). The base incorporated was confirmed by DNA sequence analysis (Figure 6). Translesional synthesis past dG-N2-AAFwas examined by steady-statekinetic analysis, using the Klenow fragment of DNA pol I (Table 11). All four DNA bases are incorporated opposite dG-N2-AAF, with dAMP being inserted preferentially opposite the lesion. The relative frequency of chain extension from the 3' primer terminus was determined using primed templates in which the lesion is paired at the 3' primer terminus with one of four DNA bases. Chain extension was detected only when dA-dGN2-AAFwas at the 3' primer terminus. The frequency of translesional synthesis past dG-N2-AAF,as estimated by Fi, X F e ~was , 10-fold less than for dG-C8-AAF (Table 111). These studies indicate that DNA polymerase I miscodes on templates containing dG-N2-AAF,selectively inserting dAMP opposite the lesion during translesional synthesis. Molecular modeling studies reveal that dG-N2-AAF (anti)can form stable pairs with dC (anti)or dA ( s y n )in B-DNA, with the aminofluorene moiety occupying the minor groove. Watson-Crick pairing is maintained in the dC*dG-N2-AAFpair; dA-dG-N2-AAFcan be stabilized by Hoogsteen pairing (Figure 7). The chromophore can be accommodated in either of two positions in which the molecule is rotated 180' around the band connecting the
Nucleotide Misincorporation by dG-N2-AAF
Chem. Res. Toxicol., Vol. 6, No. 6, 1993 823
Table 111. Frequency of Nucleotide Insertion and Chain Extension for dG-N*-AAF,dG-CS-AAF,or dG-CS-AF In Reaction Catalyzed by Klenow Fragment. C.Gb A-G C*dG-N’-AAFb A*dG-N’-AAF C*dG-CS-AAFC A*dG-C8-AAF C*dG-C8-AFC A-dG-CS-AF
Fi,
Felt
1.0 5.74 X 1 P
1.0 1.64 X 10-1
1.0 9.41 X 10-8
4.29X 1od 1.06 X
ND
ND
6.55 X le 3.78 X 1 P 0.36 1.13 X 1od
4.50 X le 1.56 X 1od
2.95 X 10-8
7.08 X
2.54 X le2 4.50 X 10-”
le
5.70 X
lo-’’
3.98 X 1o-S
Fi,
X Fext
6.04 X
5.90 X
Calculated for F i , X Fed (CeG “right” base) = 1.0. Data for G and dG-N2-AAF taken from Table 11. Data for dG-C8-MF and dG-C8-AF taken from Shibutani and Grollman (21). ND = not detectable. H
I
dA* dG-N?AAF
(syn)(anti)
Figure 7. The proposed pairing of dG-N2-AAFwith dA.
adducted base to the fluorenyl moiety. In constructing modified DNA templates for kinetic studies, a 10-mer, in which the lesion is positioned 5 bases from the 3’ terminus, was extended using the exo- Klenow fragment (Figure 2). Extension to form the full-length product was 5 times more effective when dA, rather than dC, was positioned opposite dG-N2-AAF in the primer strand. The observed preferential extension from dA-dGN2-AAFtermini is consistent with results of our kinetic studies. In the primer used in these experiments, dA is located 3’ to the position opposite dG-N2-AAF. Stacking of two purines provides a more stable 3’ terminus for further extension than when dC is positioned opposite the adduct. Coupled with a higher Fin,for dAMP, this sequence-specific factor may promote translesional synthesis past dG-N2-AAF. As shown for 8-oxoguanine, miscoding specificity of damaged DNA may be influenced by the DNA polymerase( 8 ) involved in translesional synthesis (25). DNA polymerase III* blocked DNA synthesis one base 3’ to dGN2-AAF (data not shown), suggesting that the 3’-+5’ exonucleasefunction of pol III* effectivelyremoves dNTPs inserted opposite the lesion. Miscoding observed when exo- DNA pol I copies DNA templates modified with dGN2-AAFmay be revealed in vivo when the 3‘+5’ exonuclease activity of the polymerase is suppressed, as may occur following induction of the SOS response in E. coli (34). dG-Cg-AF, the major aminofluorene adduct formed in vitro, does not miscode when copied by pol I (21)or by Pol
111*,2as detected by primer-extension studies. The level of dG-CS-AAF in tissues and cells decreases over time, reflecting endogenous DNA repair processes, while dGN2-AAFpersists in the DNA of animals treated with 2-AAF (4,6-9). The relative resistance of dG-N2-AAFto repair may reflect lack of helical distortion and/or the shielded position of the adduct in the minor groove (9,35). Thus, when considering the origins of mutations generated by 2-AAF, the potential contribution of dG-N2-AAF,dG-CSAAF, and minor adducts, such as oxidation products (30, 22),cannot be discounted. Reports of G T transversions (15,16,18,19,36),the primary class of base substitution observed in cells treated with 2-AAF, are consistent with the miscoding specificity reported for dG-N2-AAF and dG-CS-AAF adducts. The sequence used for this study was designed to explore the miscoding properties of the dG-N2-AAF. A model has been proposed in which the propensity for a particular base sequence to generate frameshift deletions at the site of DNA damage is determined by the nature of the base inserted relative to the sequence context in which the lesion is embedded and the overall efficiency of translesional DNA synthesis (21). On the basis of this model and on the observed preference for insertion of dAMP opposite dG-NZ-AAF, we predict that sequences in which T or TT are located 5’ to the lesion will lead to one-base and twobase deletions, respectively. Values for Fi, and F,,t are within the range observed for DNA adducts that generate frameshift deletions (21). Mutations may be greatly amplified during DNA replication; the so-called “minor” adducts formed in vivo, including dG-N2-AAF and dG-CS-AAF, may contribute significantly to base substitution mutagenesis by the mechanism reported in the present paper, or by sequencespecific frameshift deletions, generated by DNA template misalignment (21).
-
Acknowledgment. We are grateful to Dr. Moisbs Eisenberg for advice and assistance with molecular modeling, to Mr. Robert Rieger for preparing unmodified oligodeoxynucleotides, to Dr. Kenneth Marians for supplying DNA polymerase III*, and to Ms. Susan Rigby for typing the manuscript. This research was supported by Grant ES04068 from the National Institutes of Health.
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