Chem. Res. Toxicol. 1995,8,157-163
167
Miscoding by the Exocyclic and Related DNA Adducts 3,N4-Etheno-2’-deoxycytidine, 3,N4-Ethano-2‘-deoxycytidine, and 3-(2-Hydroxyethyl)-2’-deoxyuridine Weifeng Zhang, Francis Johnson, Arthur P. Grollman, and Shinya Shibutani” Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11 794-8651 Received July 29, 1994@
3,N4-Etheno-2’-deoxycytidine, 3-(hydroxyethyl)-2’-deoxyuridine,and 3,N4-ethano-2’-deoxycytidine are found in DNA of cells treated with either vinyl chloride or 1,3-bis(2-chloroethyl)nitrosourea. These exocyclic and related DNA adducts were incorporated into oligodeoxynucleotides, which were then used as templates for primer extension in reactions catalyzed by the Klenow fragment of Escherichia coli DNA polymerase I. The miscoding potential of each lesion was determined quantitatively. DNA primers were readily extended on an cdC-modified template; dAMP and dTMP were incorporated opposite the lesion. With high concentrations of DNA polymerase, small amounts of fully extended reaction products containing dAMP and dGMP or one-base and two-base deletions opposite ethano-dC were formed. Primer extension was blocked partially on templates containing 3-(hydroxyethyl)-dU;dAMP and smaller amounts of dTMP and dCMP were incorporated. The frequencies of nucleotide insertion opposite each of the three lesions and the frequencies of chain extension from the 3’-primer terminus, determined by kinetic analysis, were consistent with results of experiments utilizing polyacrylamide gel electrophoresis. We conclude from these studies that cdC, ethano-dC, and 3-(hydroxyethyl)-Uare potentially miscoding lesions; only cdC facilitates translesional synthesis.
Introduction Vinyl chloride is manufactured in large quantities by the chemical industry for use in the production of synthetic polymers. Occupational exposure to this volatile chemical carcinogen is associated with the development of human liver sarcoma (1-4). Following microsomal activation, vinyl chloride is converted to a-chloroethylene oxide and chloroacetaldehyde (5); these metabolites are highly mutagenic, reacting with DNA bases to form the exocyclic nucleoside adducts, cdC,l cdG, and cdA (6-8)(Figure 1). Exocyclic and related DNA adducts are also generated by treating cells with the antitumor agent 1,3-bis(2chloroethy1)nitrosourea (BCNU) (9-11). Under physiological conditions, BCNU decomposes spontaneously to form P-chloroethanediazohydroxide.This powerful alkylating agent reacts with N-3 of cytosine in DNA to generate 3-(2-~hloroethy1)-2’-deoxycytidine. The modified base then undergoes spontaneous intramolecular cyclization to form 3,N4-ethano-2’-deoxycytidine(ethano-dC) or hydrolysis to produce 3-(2-hydroxyethyl)-2’-deoxycytidine (3HE-dC). Under physiological conditions,the N-4 amino group of 3HE-dC is lost, generating 3-(2-hydroxyethyl)2’-deoxyuridine (3-HE-dU) (12, 13). These exocyclic related adducts (Figure l), along with interstrand crosslinks, are likely to be involved in the cytotoxic and carcinogenic activity of BCNU (14).
* Corresponding author.
Abstract published in Advance ACS Abstracts, December 1,1994. Abbreviations: BCNU, 1,3-bis(2-chloroethyl)nitrosourea;E&, 1JPetheno-dA; cdC, 3,iV4-etheno-2’-deoxycytidine;cdG, W,3-etheno-dG 3HE-dU, 3-(2-hydroxyethyl)ethano-dC, 3JV4-ethano-2’-deoxycytidine; dNTP, 2’-deoxyuridine; 3HE-dC, 3-(2-hydroxyethyl)-2’-deoxycytidine; 2’-deoxynucleoside triphosphate; Fm,frequency of base insertion; F d , frequency of chain extension; PAGE,polyacrylamide gel electrophore@
818.
dH
dH
dH JHEdC; X - W
cdC
SHEdU;
dhUrodC
x =0
cdG
CdA
Figure 1. Structures of some exocyclic DNA adducts.
To explore the mutagenic properties of the family of structurally-related exocyclic adducts derived from vinyl chloride and BCNU, we synthesized oligodeoxynucleotides containing a single cdC, 3HE-dU, or ethano-dC lesion (13). Base substitutions and deletions were determined quantitatively following DNA synthesis on sitespecificallymodified templates (15).Kinetic parameters of translesional synthesis were determined under steadystate conditions (16,171.Using these methods, miscoding specificities of the three lesions were compared. We observed profound differences in miscoding potential and rates of translesional synthesis.
0893-228x/95/2708-0157$09.00/00 1995 American Chemical Society
I58 Chem. Res. Toxicol., Vol. 8, No.1, 1995 Table 1. Oligodeoxynucleotidee er
S e-
5'
1 2 3 4
5 6
7 8
3' CClTCXCI'ACTTTCCI'CTCCATl" CCITCXCI'ACTTTCCTCT AGAGGAAAGT AGAGGAAAGTAG AGAGGAAAGTAGN AGAGGAAAGTAGNGAAGG AGAGGAAAGTAGGAAGG AGAGGAAAGTAGAAGG
a Base sequence of templates, primers, and standard markers. X = dC, cdC, 3-HE-dC, or ethano-dC; N = C, A, G, or T.
Experimental Procedures Materials and Methods. Organic chemicals used to synthesize oligodeoxynucleotideswere supplied by Aldrich Chemical Co., Inc. (Milwaukee, WI).HPLC grade acetonitrile, triethylamine, and distilled water were purchased from Fisher Chemical (Pittsburgh, PA). [y-32PJATP(specific activity '5000 Ci/mol) was obtained from Amersham Corp. (Arlington Heights, IL). Cloned Klenow fragments of Escherichia coli DNA polymerase 5' exonuclease I (pol I), with (exo+) and without (exo-) 3' activity, were purchased from the U S . Biochemical Corp. (Cleveland, OH), and T4 polynucleotide kinase was purchased from Stratagene (La Jolla, CA). A Waters Corp. 990 HPLC instrument, equipped with photodiode array detector, was used for separation and purification of oligodeoxynucleotides. U V spectra were measured by a Hewlett Packard 8452A diode array spectrophotometer.
-
Synthesis of Oligonucleotides Containing Exocyclic and Related DNA Adducts. DMT-phosphoramidite derivaand ethanotives of cdC, 3-[2-(benzoyloxy)ethyll-N4-benzoyl-dC, dC intermediates used in automated DNA synthesis were synthesized by methods described elsewhere (13). Modified and unmodified oligodeoxynucleotides(Table 1)were prepared with a DuPont Coder 300 automated DNA synthesizer. Oligomers were simultaneously removed from the resin support and deprotected by treatment with concentrated ammonia a t 55 "C for 3 h. Isolation was accomplished on a reverse-phase column, pBondapak CIS(0.39 x 30 cm, Waters), eluted over 60 min with a linear gradient of 0.05 M triethylammonium acetate buffer (pH 7.0) containing 10-20% acetonitrile, using a flow rate of as described previously (18).Oligodeoxynucleotides 1.0 "in, were further purified by electrophoresis on 20% polyacrylamide gel in the presence of 7 M urea. Bands were visualized under ultraviolet light and extracted overnight at 4 "C with 2.0 mL of distilled water. Extracts were concentrated on a Centricon 3 filter (Amicon)by centrifugation at 5000 rpm for 2 h. The final products were subjected to HPLC to remove urea (19). The oligomer containing 3HE-dC was hydrolyzed in aqueous solution at pH 8.0 and 37 "C for 40 h. This procedure completely converted 3HE-dC to 3HE-dU (12), as confirmed by enzymatic analysis (19) and fast atom bombardment mass spectroscopy (13). Primer Extension Reactions. Using an 18-mer template (Sequence 2,0.75 pmol) annealed to a 32P-labeled10-mer primer (Sequence 3, 0.5 pmol), primer extension reactions, catalyzed by exo- or exo+ Klenow fragments of pol I, were conducted a t 25 "C in 10 p L of a buffer composed of 50 mM Tris-HC1 (pH 8.0), 8 mM MgC12, 5 mM 2-mercaptoethanol, and four d N T P s (100 pM each) (20). Reactions were stopped by adding formamide dye and heating to 95 "C for 3 min. Samples were subjected to electrophoresis on a 20% polyacrylamide gel containing 7 M urea (35 x 42 x 0.04 cm). Bands were located by autoradiography and excised from the gel, and the radioactivity was measured by liquid scintillation counting. Certain primer extension reactions utilized a 24-mer template (Sequence 1,0.75pmol) annealed to a 32P-labeled 10-mer primer (Sequence 3, 0.5 pmol) to quantify all base substitutions and deletions. These reactions were conducted at 25 "C for 1 h in
Zhang et al. the presence of four dNTPs using exo- and exo+ Klenow fragment, as described above; however, for these experiments, reaction products were subjected to electrophoresis on two-phase 20% polyacryamide gels (15 x 72 x 0.04 cm) containing 7 M urea in the upper phase and no urea in the lower phase (15). To avoid reannealing between the templates and the reaction products in the lower phase, a 24-mer template was used instead of 18-mer template. Kinetic Studies of Nucleotide Insertion and Extension. Kinetic parameters reflecting frequencies of nucleotide insertion opposite the lesion and chain extension from the 3' terminus of the primer were established under conditions similar to those described for the primer extension assay (19). Reaction mixtures containing 0.005-0.05 unit of exo- and exo+ Klenow fragment were incubated a t 20 "C for 1-4 min in buffers containing 10 pL of Tris-HC1 (pH 8.01, using as a template Sequence 2 (1.0 pmol), primed with 0.5 pmol of a 32P-labeled 12-mer (Sequence 4) to measure base insertion, or with a 13mer (Sequence 5) to measure chain extension. Samples were subjected to 20% PAGE (35 x 42 x 0.04 cm) in the presence of 7 M urea. The Michaelis-Menten constant (K,) and maximum rates of reaction (Vmm)were obtained from Hanes-Woolf plots of the kinetic data. Frequencies of base insertion (Fins) and chain extension (Fed)were determined by equations developed by Mendelman et al. (16, 17),where F = (VmdKm)[wrongpairy (V,,/K,)[right pair], the "wrong pair" being defined as any base pair containing a single modified nucleoside and the "right pair" as d W C . 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 was extended (16).
Results Primer Extension Reactions. Primer extension reactions, catalyzed by the exo- or exo+Klenow fragment of DNA polymerase I, were conducted in the presence of four dNTPs. In reactions catalyzed by the exo- Klenow fragment, primer extension on an unmodified template produced a single fully-extended product within 5 min (Figure 2A, lane 2). On an cdC-modified template, the lesion is readily bypassed to form fully-extended products (Figure 2A, lanes 7-10). Small amounts of 19-mer bluntend addition products are observed. In contrast, primer extension on templates containing ethano-dC is blocked opposite, and one base before, the lesion (Figure 2A, lanes 17-20). Two bands are observed at the position opposite ethano-dC (13-mer position). Maxam-Gilbert sequence analysis (data not shown) demonstrates that the upper band represents a product in which dGMP is incorporated opposite the lesion while the lower band contains dAMP. Primer extension on templates containing 3HE-dU is partially blocked; two Illy-extended products are formed, representing 12%of the original primer (Figure 2A, lane 15). In reactions containing exo+ Klenow fragment, the rate and extent of primer extension past cdC and 3HEdU are slightly reduced (Figure 2B). Quantitation of Base Substitutions. Fully-extended reaction products were analyzed by two-phase gel electrophoresis (15)to determine miscoding specificities of cdC and 3HE-dU. As shown in Figure 3A (lanes 1and 6), a standard mixture of 32P-labeled oligodeoxynucleotides containing dC, dA,dG, or dT at the position of the lesion and one- or two-base deletions are completely resolved by this method. Using the exo- Klenow fragment, DNA synthesis on an unmodified template leads to the expected incorporation of dGMP opposite dC at position 13 (Figure 3A, lane 2). The full-length product represents 94% of the starting primer. When an cdCmodified template was used (Figure 3A, lane 3), dAMP
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 159
Miscoding by Exocyclic and Related DNA Adducts
---dC
edC
0 5 10 20 60
0
10 20 60 0 5 10 20 60 0
5
min
3HE-dU
ethano-dC
5 1 0 20 60
min
min
min
-
13mer l2mer -
1Omer ..
1
2
3
4
6
5
8
7
10 11 12 13 14 15 16 17 18 19 20
9
---dC
0 5
cdC
1 0 20
10 20 60 0 5
60 0 5
ethano-dC
10 20 60 0 5
mln
min
mip
lamer
3HE-dU
1 0 20
60
min
-
-
13mer 1Pmer .lOmer
1
2
3
4
5
6
7 8
9
10
11 12 13 14 15 16 17 18 19 20
Figure 2. Time course of primer extension reactions catalyzed by exo- and exo+ Klenow fragments. Using unmodified or modified 18-mer templates (Sequence 2, Table 1)primed with a 5'- 32P-labeled10-mer (Sequence 31, primer extension was determined a t 25 "C, using 0.01 unit of exo- (A) or exo+ (B) Klenow fragment for the unmodified template and 0.05 unit for the modified templates, as described under Experimental Procedures. One-third of the reaction mixture was subjected to denaturing 20% PAGE (35 x 42 x 0.04 cm).
(69%) and dTMP (19%) are incorporated opposite the lesion. Using a template containing 3HE-dU and a 10fold excess (0.5 unit) of exo- Klenow fragment (Figure 3A, lane 4), dAMP (70%) is incorporated opposite the lesion and smaller amounts of dTMP (4.5%) and dCMP (3.0%)were detected. The effect of exonucleolytic proofreading on the incorporation of bases opposite EdC and 3HE-dU was established by comparing patterns of base incorporation in reactions catalyzed by exo- and exo+ Klenow fragment (panels A and B of Figure 3). Incorporation of dTMP opposite EdC is greater in reactions catalyzed by exo+ Klenow fragment; similar amounts of dAMP (48%) and dTMP (47%)are incorporated opposite the lesion, along with a small amount of dCMP (1.8%). In reactions containing the 3' 5' exonuclease, dAMP (40%) and markedly increased amounts of dCMP (31%) are incorporated opposite 3HE-dU. Incorporation of dTMP was not observed. Fully-extended products were not found in reactions containing an ethano-dC-modified template under conditions used in the experiment shown in Figure 3; however, when increased amounts of exo- Klenow fragment were added to the reaction (Figure 4A, lane 4), small quantities of fully-extended products containing dAMP (5.4%) and dGMP (0.47%) opposite the lesion were formed; in addition, one-base (2.0%)and two-base (2.3%)deletions were observed (Figure 4B). Bands corresponding to one- and two-base deletions can be separated from products one and two bases shorter in which dCMP, dAMP, and dTMP
-
are inserted opposite the lesion. Such bands cannot be separated completely from comparable products in which dGMP is opposite the lesion. Although we confirmed the sequences of products containing deletions induced by etheno-dC (data not shown), small amounts of incomplete extended products containing dGMP opposite the lesion might be present. Kinetic Studies of Nucleotide Insertion and Extension. Steady-state kinetic parameters were established for insertion of nucleotides opposite EdC, 3HE-dU, and ethano-dC, and for chain extension from 3' termini containing these lesions using the exo- Klenow fragment. As shown in Table 2, the frequency of insertion (Fins) for dAMP opposite EdC is 3.1 times higher than for dTMP, and 47 and 225 times higher than for dGMP and dCMP, respectively. The frequency of chain extension (Fed)from 3' termini containing dA-cdC is 1.8times lower than from dT*EdCand 1order of magnitude higher than from other base pairs. Thus, the overall frequency of translesional synthesis (Fins x Fed) when dAMP is inserted opposite EdC is 1.7-fold higher than for dTMP insertion and 4104200 times higher than for other nucleotides. Fin, for dAMP opposite ethano-dC is 3.3 times higher than for dGMP; however, Fed from dA*ethano-dCis 1.5 times lower than from dGethano-dC. Thus, the overall frequency of translesional synthesis past ethano-dC is only 2.1-fold higher for dAMP than for dGMP, but 3 orders of magnitude higher than for other nucleotides. The Fin, of dAMP opposite ethano-dC is similar to its F i n s opposite EdC, but Fed from dA*ethano-dCtermini is much
Zhang et al.
160 Chem. Res. Toxicol., Vol. 8, No. 1, 1995 (A)
ethano-dC 0.1
0.5
1.0 5.0 (units)
Stn.
-
18A - i8C -17A'
r*
-1%
- 16A'
-18A -18T
-17A' -13G -13A
- 18C
- 12 1
(6)
2
Stn.
dC
5
4
3
EdC
6
3HE-dU ethano-dC
Stn.
1
2
3
4
Figure 4. Nucleotide incorporation opposite ethano-dC lesion
18G 18A -
as a function of DNA polymerase concentration. (A) Using the ethano-dC-modified 24-mer template (Sequence 1, Table 1) primed with a 32P-labeled10-mer (Sequence 31, primer extension reactions, catalyzed by exo- Klenow fragment, were conducted for 1h at 25 "C. One-third of the reaction mixture was subjected to denaturing 20% PAGE (35 x 42 x 0.04 cm). (B) One-third of the sample used for the experiment shown in lane 4 of panel A was subjected to two-phase 20% PAGE (15 x 72 x 0.04 cm). Lanes 5 and 7 represent a mixture of 18-mer standard markers (sequences6-8) containing dC, dA,dG, or dT opposite the lesion and/or one-base (A1) or two-base (A2) deletions.
18T -
17d-
18C 16A*-
1
2
3
4
5
6
Figure 3. Nucleotide incorporation opposite EdC, SHE-dC, and ethano-dC lesions in reaction catalyzed by exo- and exo+Klenow fragments. Using a 24-mer template (Sequence 1 in Table 1) primed with a 5'-32P-labeled 10-mer (Sequence 3), the primer was allowed to extend for 1h at 25 "C, using 0.01 units of exo(A) or exo+ (B) Klenow fragment for the unmodified template. 0.05 unit of exo- Klenow fragment (A) or 0.1 unit of the exo+ Klenow fragment (B) for the EdC modified template and 0.5 unit for 8HE-dU- and ethano-dC-modified templates as described under Experimental Procedures. One-third of the reaction mixture was subjected to two-phase 20% polyacrylamide gel electrophoresis (15 x 72 x 0.04 cm). Mobilities of reaction products were compared with those of 18-mer standards (Sequences 6-8) containing dC, dA,dG, or dT opposite the lesion and one-base (A1) or two-base (A2) deletions a t the position of the lesion (A and B, lanes 1 and 6).
lower than from dA-cdC. Thus, the relative amount of fully-extended products produced on templates containing ethano-dC should be much less than on templates containing EdC, as reflected in the PAGE analysis of primer extension experiments (Figure 2A). On a 3HEdU-modified template, Fin, for dAMP and Fed from the dA.3HE-dU terminus are higher than for other nucleotides. The estimated frequency of translesional synthesis past this lesion follows the order: dAMP =- dTMP > dCMP, dGMP. Fins for dAMP opposite 3HE-dU and Fed from a 3' terminus containing dA-3HE-dU are 1.9 times and 2.9 times lower than for comparable reactions involving cdC. Fins x Fed for dAMP paired with 3HEdU is 5.6 times lower than when paired with EdC. These kinetic data are consistent with results of PAGE analysis (Figure 2).
Discussion The miscoding potential of EdC was determined in primer extension reactions containing purified Klenow
fragment of DNA polymerase I and equimolar amounts of four dNTPs. A recently-developed PAGE system (15) was used to quantify base substitutions and deletions in vitro. dAMP and dTMP are incorporated preferentially opposite EdC; DNA synthesis past this lesion was relatively complete. These observations are supported by steady-state kinetic analysis in which nucleotide insertion opposite EdC and extension from the 3'-primer terminus are measured in the presence of a single dNTP. The Fins for dAMP opposite EdC is 3.1 times higher than for dTMP; insertion frequencies for both nucleotides are higher than for dGMP or dCMP. These results are consistent with the time course of dNMP incorporation opposite EdC as reported by Simha et al (21), using the exo- Klenow fragment of DNA polymerase I. Fed from 3' termini containing dT*cdCand dA*EdCis much higher than for the other two pairs and 1.8 times and 1.4 times higher when exo- (Table 2) and exo+ (data not shown) Klenow fragments are used to catalyze the reaction. These data differ from those reported by Simha et al. (21) who reported that chain extension occurs frequently only when dT is paired with EdC. The Fins for a given dNTP, coupled with the Fed, represent a kinetic parameter that has been used successfully to predict miscoding by DNA lesions during translesional synthesis (22). The frequency of translesional synthesis, Fins x Fed, past EdC follows the order: dAMP > dTMP >> dGMP > dCMP. The value for dAMP is 1.7-fold higher than for dTMP. These values are supported by the observation that primer extension on templates containing cdC produces fully-extended products containing dAMP and dTMP opposite the lesion. Using the exo+ Klenow fragment (data not shown), Fed for dAvC (2.85 x 10-l) was similar to that of exo- Klenow fragment (3.74 x 10-l). The proofreading function may not work when dA was paired to EC. Thus, the miscoding properties of cdC, as established with the Klenow fragment of DNA pol I, predict C T transitions and C A
-
-
Chem. Res. Toxicol., Vol. 8, No. 1, 1995 161
Miscoding by Exocyclic and Related DNA Adducts
Table 2. Kinetic Parameters for Nucleotide Insertion and Chain Extension Reactions Catalyzed by the exo- Klenow FragmenP insertion:
extension: dGTP 1NG--32P 13-mer
dNTP 1G--3aP 12-mer
5'-CcTfCxC--
5'-CcITCxC--
K, (uM) 1.5 f 0.2
N-X
G.c
V,, (% min-l) 59.8 f 1.8
K m &MI 4.6 f 0.2
Vm, (% min-l) 7.90 f 0.5
1.58 x 3.56 x 7.53 x 1.16 x
36.5 & 4.1 13.5 f 1.6 31.5 f 2.3 16.3 f 1.2
Fin. 1.0
1.0
F&
Fins x Fext 1.0
1.25 f 0.03 8.67 f 0.62 2.32 f 0.31 19.0 f 3.1
1.99 x 3.74 x lo-' 4.29 x 6.79 x 10-1
3.14 x 1.33 x 3.23 x 7.88 x
X = EdC
c.x
140 f 6.0 8.8 f 0.5 41.0 i 4.0 22.0 i 3.0
0.9 f 0.1 12.5 f 3.2 1.2 f 0.11 10.2 f 0.5
c*x
106 f 6.1 10.8 f 0.9 68.8 f 5.6 38.0 f 0.9
8.9 f 1.0 8.0 f 0.5 4.2 f 0.4 3.9 f 0.4
2.11 10-3 1.87 x 1.53 x 2.55 x lov3
45.5 f 2.1 18.2 f 1.3 49.0 f 3.7 42.1 f 4.3
1.05 f 0.11 3.96 f 0.11 1.52 f 0.11 3.24 f 0.25
1.34 x 1.27 x 10-l 1.81 x 4.48 x
2.83 x 2.37 x 2.77 x 1.14 x
c*x
130 f 11 12.2 f 1.1 21.2 f 2.7 86.2 f 7.7
1.5 f 0.1
2.89 x 1.81 x 5.48 x 2.94 x
45.2 f 0.6 13.3 f 1.8 21.5 f 1.7 39.6 f 4.7
0.11 f 0.01 0.22 f 0.03 0.55 f 0.03 0.21 f 0.03
1.42 x 9.63 10-3 1.49 x 3.09 x 10-3
4.10 x lo-' 1.74 10-4 8.17 x 9.08 x 10-7
A-X GX T-X X = SHE-dU A*X GX T.X X = ethano-dC A*X GX T.X
8.8 f 0.3
4.6 f 0.1 1.0 f 0.1
a Kinetics of insertion and extension reactions were determined as described under Experimental Procedures. Frequencies of nucleotide insertion (Fins)and chain extension (Fea)were estimated by the equation: F = (Vma,/K,)[wrongpairY(V,exlK,)[right pair], where "right pair" = dGdC. X = lesion.
B)
A)
NOAT-8
'
N-A
~
~
1
AG--AT-
6'cc=x?-
~
NGAT~'CCTTCXCTA-~ ~ ~ N=G
~
-
1
G-GAT"CCTTC, PTAX
Figure 6. Mechanism for one- and two-base deletions produced by ethano-dC. X = ethano-dC.
transversions. Although the Klenow fragment is not a replicative enzyme, its miscoding properties are consistent with reports of the mutagenic specificity of this lesion in E. coli (23-25) and mammalian cells (25). Ethano-dC differs structurally from cdC in that the exocyclic ring is partially saturated. Primer extension reactions on ethano-dC-modified templates are blocked opposite and one base 3' to the lesion while cdC is readily bypassed. Furthermore, the two lesions exhibit quite different miscoding properties. When large amounts of exo- Klenow fragment were used, small amounts of fullyextended products containing dAMP and dGMP opposite ethano-dC were detected, accompanied by products containing one- and two-base deletions at the site of the lesion. The frequency of translesional synthesis past ethano-dC is at least 2 orders of magnitude lower than for cdC and follow the order: dAMP > dGMP >> dTMP > dCMP. These kinetic parameters are consistent with the general mechanism proposed for generation of frameshift deletions in which the propensity for template misalignment was shown to depend on (a) sequence context, (b) nature of the base inserted opposite the lesion, and (c) the frequency of translesional synthesis (22). Thus, in Sequence 1, when dAMP is incorporated opposite ethano-dC, the newly inserted base pairs with dT two positions 5' to the lesion to form a two-base deletion (Figure 5A). Similarly, when dGMP is incorporated opposite the lesion, the newly inserted base pairs with dC 5' to the lesion to form a one-base deletion (Figure 5B).
-
Using a 3HE-dU-modified template and exo- Klenow fragment, dAMP is incorporated preferentially opposite the lesion. Small amounts of dTMP and dCMP also are incorporated. When the exo+Klenow fragment was used, increased amount of dCMP were incorporated opposite the lesion. Feb from dC-3HE-dUtermini is 10 times lower than from dA.3HE-dU in reactions containing the exofragment; however, with exo+ Klenow fragment, Fee for dC.3HE-dU (3.59 x 10-9 and dA.3HE-dU (3.30 x 10-9 are similar (data not shown). Thus, the exonucleolytic proofreading function may preferentially remove dAMP when paired to 3HE-dU. In the absence of a specific repair mechanism for 3HE-dU, the miscoding properties of this lesion, as established in this study with Klenow fragment, would lead to C T transitions and, to a lesser extent, C A and C G transversions. Mutational spectra of BCNU have been reported for mammalian cells. G -. T transversions predominate, followed by G A transitions (26, 27). These spectra are thought to reflect 06-alkylation predominantly by metabolites of this drug and differ therefore from those induced by ethano-dC and 3HE-dU. The blocking effects of ethano-dC and 3HE-dU may reduce their contribution to mutagenicity in vivo; also, exocyclic lesions in DNA may be effectively repaired in cells (28,29). The exocyclic imidazole ring of cdC is expected to be planar and to generate a relatively hydrophobic environment. These properties allow the exocyclic ring to stack with neighboring bases during DNA synthesis. Since formation of the exocyclic imidazole ring of cdC involves N-3 and N-4, GdC cannot adopt Watson-Crick base pairing with dG. Four possible base-pairing arrangements for cdCdA and cdCdT pairs are shown in Figure 6. When paired with dA,a hydrogen bond can stabilize the complex only when cdC adopts the anti conformation. A single hydrogen bond also can form between dT and either the anti or syn form of cdC. Thus, the possibilities for stabilization of pairs between cdC and dA,or between cdC and dT, are limited. Nevertheless, cdC promotes rapid incorporation of dAMP and dTMP opposite the lesion. The exocyclic ring of ethano-dC is slightly distorted from the planar conformation; however, hydrogen atoms
--
-
-
162 Chem. Res. Toxicol., Vol. 8, No. 1, 1995
Zhang et al.
cdC (syn) : dA
cdC (anti) : dA
cdC (syn) : d f
cdC (anti) : dT
H
JHE-dU : dA
3HE-dU : dC
Figure 6. Possible base-pairing arrangements for EdC and 3HE-dU.
at the C7 and C8 positions of this lesion are unlikely to interfere markedly with base stacking. Rather, factors that account for misincorporation of bases opposite cdC and the blocking effect of ethano-dC during DNA synthesis may have their origin in the electronic nature of these two lesions. Alkylation of deoxycytidine at N-3 is known to increase the basicity of the N4 nitrogen atom, as reflected in the change in pKa from 4.3 for dC to 8.8 for 3-(Me)dC(12,301.Using spectrophotometric titration, we have found the pKa of ethano-dC to be 8.9, indicating that, under physiological conditions, the heterocyclic base is -90% protonated. This will change the dipole of the molecule significantly, thus affecting stacking and causing the base to behave as a hydrogen donor at N4 rather than as a hydrogen acceptor. Two hydrogen bonds can be formed when 3HE-dU is paired with dA or dC (Figure 6); nevertheless, translesional synthesis past 3HE-dU is much less efficient than past cdC. Again, no direct relationship can be observed between the efficiency of translesional synthesis and the number of potential hydrogen bonds. Thus, exogenous and endogenous factors (31),other than base pairing, may determine patterns of nucleotide incorporation and the efficiency of DNA synthesis past this lesion. Acknowledgment. We thank Mr. R. Rieger and Ms. M. C . Torres for synthesizing oligodeoxynucleotides and Dr. Jerry Solomon for drawing our attention to the hydrolytic deamination reaction of hydroxycytosine. This work was supported in part by NIH Grants CA17395 and CA47995 to A.P.G.
References (1) Creech, J. L., Jr., and Johnson, M. N. (1974) Angiosarcoma of liver in the manufacture of polyvinyl chloride. J . Occup. Med. 16, 150-151. (2) Maltoni, C., and Lefemine, G. (1974) Carcinogenicity bioassays of vinyl chloride. Enuironm. Res. 7 , 387-450. (3) Merletti, F., Heseltine, E., Saracci, R., Simonato, L., and Wilboum, J. (1984) Target organs for carcinogenicity of chemicals and
industrial exposures in humans: a review of results in the IARC monographs on the evaluation of the carcinogenic risks to humans. Cancer Res. 44,2244-2250. (4) IARC (1979)Vinyl chloride, polyvinyl chloride and vinyl chloridevinyl acetate copolymer. In IARC monographs on the evaluation of the carcinogenic of chemicals to humans, pp 348-377, IARC, Lyon. (5) Barbin, A., Besson, F., and Perrard, M. (1985) Induction of specific base-pair substitutions in E. coli. trpA mutants by chloroethylene oxide, a carcinogenic vinyl chloride metabolite. Mutat. Res. 152, 147-156. (6) Barbin, A., and Bartsch, H. (1986) Mutagenic and promutagenic properties of DNA adducts formed by vinyl chloride metabolites. In The role of cyclic nucleic mid adducts in carcinogenesis and mutagenesis (Singer, B., and Bartsch, H., Eds.) IARC Scientific Publications 70, pp 345-358, W E C , Lyon, France. (7) Scherer, E., Van Der Laker, C. J., Gwinner, L. M., and Laib, R. J. (1981) Modification of deoxyguanosine by chloroethylene oxide. Carcinogenesis 2, 671-677. (8)Malaveille, C., Bartsch, H., Montesano, R., Barbin, A., and Camus, A. M. (1975)Mutagenicity of vinyl chloride, chloroethylene oxide, chloroacetaldehyde and chloroethanol. Biochem. Biophys. Res. Commun. 63,363-370. (9) Tong, W. P., Kohn, K. W., and Ludlum, D. B. (1982)Modifications of DNA by different haloethylnitrosoureas. Cancer Res. 42,44604464. (10) Ludlum, D. B. (1986) Formation of cyclic adducts in nucleic acids by the haloethylnitrosoureas. In The role of cyclic nucleic acid adducts in carcinogenesis and mutagenesis (Singer, B., and Bartsch, H., Eds.) IARC Scientific Publications 70, pp 137-146, IARC, Lyon, France. (11) Montgomery, J. A. (1981) The chemistry of the nitrosoureas. In Nitrosoureas in cancer treatment (Serrou, B., Schein, P. S., and Imbach, J.-L., Eds.), pp 13-20, ElsevierNorth-Holland Biomedical Press, New York. (12) Solomon, J., and Segal, A. (1989) DNA adducts of propylene oxide and acrylonitrile epoxide: Hydrolytic deamination of 3-alkyl-dCyd to 3-alkyl-dUrd. Enuiron. Health Perspect. 81, 19-22. (13) Zhang, W., Rieger, R., Iden, C., and Johnson, F. (1995) Synthesis of 3,N4-etheno, 3,iV4-ethano, and 3-(2-hydroxyethyl)derviatives of 2'-deoxycytidine and their incorporation into oligomeric DNA. Chem. Res. Toxicol. 8, 148-156. (14) Ludlum, D. B. (1990) DNA alkylation by the haloethylnitrosoureas: Nature of modifications produced and their enzymatic repair or removal. Mutat. Res. 233, 117-126. (15) Shibutani, S. (1993) Quantitation of base substitutions and deletions induced by chemical mutagens during DNA synthesis in vitro. Chem. Res. Toxicol. 6, 625-629. (16) Mendelman, L. V., Petruska, J., and Goodman, M. F. (1989) Nearest neighbor influences on DNA polymerase insertion fidelity. J . Biol. Chem. 264, 14415-14423. (17) Mendelman, L. V., Petruska, J., and Goodman, M. F. (1990) Base mispair extension kinetics. J . B i d . Chem. 265, 2338-2346. (18)Shibutani, S., Gentles, R., Johnson, F., and Grollman A. P. (1991) Isolation and characterization of oligodeoxynucleotidescontaining dG-W-AAF and oxidation products of dG-C8-AF. Carcinogenesis 12, 813-818. (19) Shibutani, S., Bodepudi, V., Johnson, F., and Grollman, A. P. (1993) Translesional synthesis on DNA templates containing 8-0~0-7,8-dihydrodeoxyadenosine. Biochemistry 32, 4615-4621. (20) Shibutani, S., Takeshita, M., and Grollman, A. P. (1991)Insertion of specific bases during DNA synthesis past the oxidationdamaged base 8-oxodG. Nature 349, 431-434. (21) Simha, D., Palejwala, V. A., and Humayun, M. Z. (1991) Mechanisms of mutagenesis by exocyclic DNA adducts. Construction and in vivo template characteristics of an oligonucleotide bearing a single site-specific ethenocytosine. Biochemistry 30, 8727-8735. (22) Shibutani, S., and Grollman, A. P. (1993) On the mechanism of frameshift (deletion) mutagenesis in vitro. J. Biol. Chem. 268, 11703-11710. (23) Basu, A. K., Wood, M. L., Niedernhofer, L. J., Ramos, L. A., and Essigmann, J. M. (1993) Mutagenic and genotoxic effects of three vinyl chloride-induced DNA lesions: 1,N6-ethenoadenine, 3,N4Biochemethenocytosine, and 4-amino-5-(imidazol-2-yl)imidazole. istry 32, 12793-12801. (24) Palejwala, V. A., Simha, D., and Humayun, M. Z. (1991) Mechanism of mutagenesis by exocyclic DNA adducts. Transfection of M13 viral DNA bearing a site-specific adduct shows that ethenocytosine is a highly efficient recA-independent mutagenic noninstructional lesion. Biochemistry 30, 8736-8743. (25) Moriya, M., Zhang, W., and Grollman, A. P. (1994) Mutagenic potency of 3,N4-ethenocytosine,a DNA lesion produced by vinyl chloride, is strikingly different in simian kidney cells and Es(in press). cherichia coli. Proc. Natl. Acad. Sci. U.S.A.
Miscoding by Exocyclic and Related DNA Adducts (26) Minnick,D. T., Veigl, M. L., and Sedwick,W. D. (1992) Mutational specificity of 1,3-bis-(2-chlomethyl)-l-nitrosoureain a Chinese hamster ovary cell line. Cancer Res. 62,4688-4695. (27) Minnick, D. T., Gerson, S. L., Dumenco, L. L., Veigl, M. L., and Sedwick, W. D. (1993) Specificity of bischlomethylnitrosoureainduced mutation in a Chinese hamster ovary cell line transformed to express human 06-alkylguanine-DNAalkyltransferase. Cancer Res. 63, 997-1003. (28) Matijasevic, Z., Sekiguchi, M., and Ludlum, D. B. (1992) Release of iP,3-ethenoguanine from chloroacetaldehyde-treatedDNA by Escherichia coli 3-methyladenine DNA glycosylase 11.Proc. Natl. Acad. Sci. U S A . 89,9331-9334.
Chem. Res. Toxicol., Vol. 8, No.1, 1995 163 (29) Dosaqjh, M. K,Chenna, A., Kim, E., E’raenkel-Conrat, H., Simson, L., and Singer, B. (1994) All four known cyclic adducts formed in DNA by the vinyl chloride metabolite chloroacetaldehyde are released by a human DNA glycosylase. Proc.Natl. h a d . Sei. U S A . 91, 1024-1028. (30) Sowers, L., Sedwick, W. D., and Shaw, B. (1989) Hydrolysis of Ns-methyl-2’-deoxycytidine:model compound for reactivity of protonated cytosine residue in DNA. Mutat. Res. 216,131-138. (31) Echols, H., and Goodman, M. F. (1991) Fidelity mechanisms in DNA replication. Annu. Rev. Biochem. 80,477-511.
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