Chem. Res. Toxicol. 1998, 11, 311-316
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Polymerase Blockage and Misincorporation of dNTPs Opposite the Ethylene Dibromide-Derived DNA Adducts S-[2-(N7-Guanyl)ethyl]glutathione, S-[2-(N2-Guanyl)ethyl]glutathione, and S-[2-(O6-Guanyl)ethyl]glutathione† Mi-Sook Kim§ and F. Peter Guengerich* Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146 Received November 18, 1997
The carcinogen ethylene dibromide (EDB) has been shown to cause glutathione (GSH)dependent base-substitution mutations, especially GC to AT transitions, in a variety of bacterial and eukaryotic systems. The known DNA adducts S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S-[2-(O6-guanyl)ethyl]GSH were individually placed at a site in a single oligonucleotide. Polymerase extension studies were carried out using Escherichia coli polymerase I exo- (Klenow fragment, Kf-) and polymerase II exo- (pol II-), bacteriophage T7 polymerase exo-, and human immunodeficiency virus-1 reverse transcriptase in order to characterize misincorporation events. Even though extension was not as efficient as with the nonadducted template, some fully extended primers were observed with the template containing S-[2-(N7-guanyl)ethyl]GSH using all of these polymerases. dCTP was the most preferred nucleotide incorporated opposite S-[2-(N7-guanyl)ethyl]GSH by most of polymerases examined; however, dTTP incorporation was observed opposite S-[2-(N7-guanyl)ethyl]GSH with pol II-. Both S-[2-(N2-guanyl)ethyl]GSH and S-[2-(O6-guanyl)ethyl]GSH strongly blocked replication by all polymerases. Only dATP and dGTP were incorporated opposite S-[2-(N2-guanyl)ethyl]GSH by both Kf- and pol II-. S-[2-(O6-Guanyl)ethyl]GSH was shown to strongly code for dATP incorporation by Kf-. With pol II-, dTTP was incorporated opposite S-[2-(O6-guanyl)ethyl]GSH. In conclusion, all three GSH-guanyl adducts derived from the carcinogen EDB blocked the polymerases and were capable of miscoding.
Introduction 1
Ethylene dibromide (EDB) has been shown to induce tumors in rodents at a number of sites (1-5). The human carcinogenicity of low-level EDB exposure is unknown, but two workers died shortly following acute exposure to EDB (6). Because of concern about toxicity and carcinogenicity, industrial use of EDB (soil fumigant, scavenger in leaded gasoline) was considerably curtailed in the 1980s (7). Bacteriophage M13mp18 was treated with S-(2-chloroethyl)GSH, an analogue of the activated form of EDB, and used to transfect Salmonella typhimurium; DNA sequence analysis of the lac Z R-complementation mutants revealed that GC to AT transitions were dominant (8). The same mutational changes (GC to AT base †
This research was supported in part by United States Public Health Service Grant R35 CA44353 and Grant P30 ES00267. M.-S. Kim was supported in part by a Merck predoctoral fellowship. * Address correspondence to this author. Tel: (615) 322-2261. Fax: (615) 322-3141. E-mail:
[email protected]. § Current address: Merck & Company, P.O. Box 2000, 126 East Lincoln Ave., Rahway, NJ 07065-0900. 1 Abbreviations: EDB, ethylene dibromide; MOPS, 3-(N-morpholino)propanesulfonic acid; Kf-, Klenow fragment (of Escherichia coli polymerase I) exo-; pol II-, polymerase II exo-; T7-, bacteriophage polymerase T7 exo- (thioredoxin mixture); HIV RT, human immunodeficiency virus-1 reverse transcriptase. The standard abbreviations for nucleic acid bases are defined in the current Instructions to Authors (see the January issue of Chem. Res. Toxicol.).
Chart 1. Structures of Guanyl Adducts Derived from EDB
transitions) have also been observed in very different biological systems such as Drosophila melanogaster, Chinese hamster ovary cells, and Escherichia coli after treatment with EDB, indicating that the biology related to EDB adducts may be rather similar among species (911). S-[2-(N7-Guanyl)ethyl]GSH was characterized as the major DNA adduct derived from EDB, and S-[2-(N2guanyl)ethyl]GSH, S-[2-(O6-guanyl)ethyl]GSH, and S-[2(N1-adenyl)ethyl]GSH are minor adducts (Chart 1) (8, 12-15). Oligonucleotides containing S-[2-(N7-guanyl)ethyl]GSH have been characterized by physicochemical methods
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(16-18). Base pairing of S-[2-(N7-guanyl)ethyl]GSH with Cyt in the complementary sequence was shown to be quite disrupted; however, the basic B-type helical structure was not perturbed (16). Comparative studies of oligonucleotides containing various N7-guanyl adducts indicated some interactions between the GSH side chain and the DNA bases (17). Dramatic variations of the ratio of S. typhimurium TA100 base-pair mutations to bacterial DNA N7-guanyl adducts were also observed (19). The frequency of O6-alkylGua lesions in DNA has been shown to be strongly correlated with mutation induction in different target genes in bacteria and cultured cells and to be critical in tumorigenesis of many chemical carcinogens (20-22). GC to AT transition is the predominant type of mutation caused by O6-alkylGua (23-27). Some N2-guanyl adducts have been shown to miscode (28, 29). We developed methods for the synthesis of oligonucleotides containing the EDB-derived DNA adducts S-[2-(N7guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, and S[2-(O6-guanyl)ethyl]GSH at a single site (30), in a sequence previously demonstrated to show mutations when M13mp18 DNA was treated with S-(2-chloroethyl)GSH, an analogue of the activated form of EDB (8). In this study, the blocking and miscoding potentials of each Gua adduct in the oligonucleotide were investigated using four model DNA polymerases in order to characterize the mechanism by which EDB causes mutations.
Experimental Procedures Chemicals. [γ-32P]dATP was purchased from DuPont NEN (Boston, MA). dNTPs were from Pharmacia (Piscataway, NJ). DNA sequencing systems (Maxam-Gilbert procedure) were from DuPont Biotechnology Systems (Boston, MA) and Sigma Chemical Co. (St. Louis, MO). Enzymes. T4 polynucleotide kinase was purchased from United States Biochemical (Cleveland, OH). DNA polymerases were purified by L. L. Furge, Department of Biochemistry, from E. coli using stock plasmids provided (280 values used in parentheses): Kf- (Klenow fragment of E. coli polymerase I, exo-) (Dr. C. Joyce, Yale University, New Heaven, CT; 280 ) 6.32 × 104 M-1 cm-1), pol II- (E. coli polymerase II exo-) (Prof. M. F. Goodman, University of Southern California, Los Angeles, CA; 280 ) 1.24 × 105 M-1 cm-1), T7- (bacteriophage polymerase T7 exo-; abbreviation T7- also refers to thioredoxin mixture) (280 ) 1.44 × 105 M-1 cm-1) and thioredoxin (280 ) 1.37 × 104 M-1 cm-1) (Prof. K. A. Johnson, Pennsylvania State University, University Park, PA), HIV RT (human immunodeficiency virus-1 reverse transcriptase) (Dr. S. Hughes, Frederick Cancer Facility, Frederick, MD; 280 ) 2.61 × 105 M-1 cm-1) (31, 32). Concentrations of diluted solutions were estimated by A280 measurements using a modified Cary 14/OLIS spectrophotometer (On-Line Instrument Systems, Bogart, GA). The ratio of T7 DNA polymerase to thioredoxin used in this study was 1:20. Polymerase Assays. General. The synthesis and characterization of the three modified templates have been described (30). The 13-mer primer d(5′-CTCGGTACCCTTG-3′) (2 mM) was 5′-end-phosphorylated with 32P using T4 polynucleotide kinase and [γ-32P]dATP and purified on a Biospin column (BioRad, Hercules, CA). Template and primer (2:1 molar ratio) were annealed in a buffer containing 50 mM sodium MOPS [3-(Nmorpholino)propanesulfonic acid] (pH 7.0), bovine serum albumin (50 µg mL-1), and 5 mM MgCl2 by incubating for 2 h at 16 °C. Concentrations of oligonucleotides were estimated using A260 measurements, with calculation of extinction coefficients as described (33): primer 260 ) 8.54 × 104 M-1 cm-1, template 260 ) 1.74 × 105 M-1 cm-1. The extended products were analyzed using a PhosphorImager system (model 400E, Molecular Dynamics, Sunnyvale, CA) and the manufacturer’s software.
Kim and Guengerich Chart 2. Oligonucleotidesa
a G* ) Gua, S-[2-(N7-guanyl)ethyl]GSH, S-[2-(N2-guanyl)ethyl]GSH, or S-[2-(O6-guanyl)ethyl]GSH. The site of first incorporation is indicated with an arrow.
Primer Extension. Primer extension reactions were performed by reacting primer/template pairs (100 nM) (Chart 2) with a mixture of dNTPs (100 µM each) in 5 µL of 50 mM sodium MOPS buffer (pH 7.0) containing 8 mM MgCl2, 4 mM dithiothreitol, and bovine serum albumin (2 µg mL-1) with each of three different concentrations of polymerases. These reactions were carried out for 1 h at 25 °C with all polymerases, except for pol II- which was incubated at 37 °C.2 Reactions were quenched by adding 5 µL of 10 mM EDTA in 90% formamide (v/v), and the products were analyzed on 20% (w/v) denaturing polyacrylamide gels, prepared using Sequagel (National Diagnostics, Atlanta, GA). One-Base Incorporation. Primers (13-mer) were extended using unmodified and adducted templates (100 nM) (Chart 2) in the presence of single dNTPs (100 µM). Preliminary reactions were performed at 25 °C for 1 h with all polymerases (except for pol II-, which was incubated at 37 °C),2 and the concentrations of polymerase used were 200 nM (Kf-), 400 nM (pol II-), 400 nM (T7), and 200 nM (HIV RT). Conditions were adjusted accordingly for steady-state kinetics (vide infra). Steady-State Kinetics. Primer extension reactions were performed according to the general procedure described elsewhere (31, 32, 35), using adducted templates in the presence of several different concentrations of single dNTPs as indicated in the table of the results. In all cases, extension was 105
kcat (min-1)
Km (µM)
21c
0.11
0.072 0.083 0.75d 0.63e
760 770 0.24 340
0.043i 0.0013k 1.8 × 10-4 m 2.0 × 10-4 o
kcat/Km
misinsertion ratioa
6 × 10-4
4400 440 0.54
190 105. dTTP incorporation and dCTP incorporation opposite S-[2-(O6-guanyl)ethyl]GSH were both detectable with pol II-. The misinsertion ratio for dTTP was 0.13, which was lower than that reported for dTTP opposite O6-methylGua with Kf- in a different sequence (1-2.5) (27). In considering the results of the polymerase kinetic experiments, comparisons can be made not only of the misinsertion ratios but also of the enzyme efficiencies (kcat/Km) for each pairing. With regard to pairing of Gua and its derivatives with dTTP by pol II-, the efficiency is ∼17-fold better for S-[2-(N7-guanyl)ethyl]GSH than Gua but the efficiencies for S-[2-(O6-guanyl)ethyl]GSH and Gua are similar (Table 1). With regard to insertion of dATP and dGTP, the efficiencies are less with both the N2- and O6-adducts than with Gua for both Kf- and pol II-. The favorable misinsertion ratios are driven not by favorable pairing of dATP and dGTP with the adducts but by the very disfavored incorporation of dCTP opposite these adducts. The molecular basis of this disfavored incorporation of dCTP (as much as 1011) may be related to the difficulty in developing canonical Watson-Crick base pairs with dCTP due to the presence of bulky N2and O6-guanyl adducts. Alternative pairing modes (e.g., wobble) may be nearly as feasible for these derivatives as for Gua itself. Exactly why the N7-adduct is a block (Table 1, Figure 1) is not clear, since the base modification is not in the base-pairing region and only changed the ∆∆G° for binding to a complemetary C by 1.4 kcal mol-1 (18). However, the GSH moiety does present considerable bulk in the major groove [when the DNA is double-stranded (16)] and may interact directly with the polymerase. Abril et al. (43) reported decreased mutations caused by EDB in bacterial strains devoid of O6-alkyltransferases. The basis of this finding is not clear, because if the O6-alkylGua lesion is mutagenic, the transferases might repair this lesion and lower the mutation rate. Possible speculation on this finding is that a repair enzyme might bind to S-[2-(O6-guanyl)ethyl]GSH without repairing it, and mutation could somehow be enhanced
Chem. Res. Toxicol., Vol. 11, No. 4, 1998 315
by a complex of DNA lesion and repair enzyme. However, nucleotide excision repair does seem to be involved and lowers the mutation frequency in experiments in which DNA is modified with the half-mustard (2-chloroethyl)GSH (8). It is not known whether S-[2-(O6-guanyl)ethyl]GSH is a substrate forsor an inhibitor ofsO6alkyltransferase. There was no detectable incorporation of any base opposite S-[2-(O6-guanyl)ethyl]GSH or S-[2(N2-guanyl)ethyl]GSH with T7- or HIV RT (Table 1), both of which are replicative polymerases, indicating that the fates of replication of EDB-guanyl adducts are highly dependent on DNA polymerases. In conclusion, the miscoding potentials of three known EDB-guanyl adducts were investigated. S-[2-(N7-Guanyl)ethyl]GSH was found to be a replication block and miscoded to insert dTTP, with pol II-, at a low frequency. S-[2-(N2-Guanyl)ethyl]GSH was shown to be capable of blocking polymerization and miscoding, with dATP and dGTP being the most preferred bases incorporated opposite this adduct. S-[2-(O6-Guanyl)ethyl]GSH was also found to be a replication block and to code for dATP and dGTP incorporation with Kf- and for dTTP incorporation with pol II-. In considering the GC to AT transition observed in many biological systems, the expected pairing of a Gua adduct with dTTP was observed only with pol II- and two adducts, S-[2-(N7-guanyl)ethyl]GSH and S-[2(O6-guanyl)ethyl]GSH (Table 1). The O6-adduct was 2 orders of magnitude more likely to miscode than the N7adduct, but the N7-adduct is present at levels at least 2 orders of magnitude higher than the O6-adduct (8). A quantitative assessment of the ability of pol II- to extend the two adducts has not been made. The in vitro assay system we used in this study is not necessarily representative of the prokaryotic cellular replication system, in which DNA polymerase III is the major replicative enzyme [prior work suggests that the SOS system is not obligatory for mutagenesis in bacteria (19)]. This study has provided useful information about how these adducts can behave with DNA polymerases, even though the information about which guanyl adduct is primarily responsible for the dominant GC to AT transitions seen in vivo is still not complete.
Acknowledgment. We thank Dr. S. Langoue¨t for valuable suggestions and L. L. Furge for purification of the polymerases used in this study and for comments on the manuscript. Supporting Information Available: Figure showing nucleotide sequence analysis of extended primers (1 page). Ordering information is given on any current masthead page.
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