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Chem. Res. Toxicol. 1996, 9, 1350-1354
Using UvrABC Nuclease To Detect 7,12-Dimethylbenz[a]anthracene anti-Diol Epoxide-DNA Binding Specificity in the Mouse H-ras Gene James X. Chen,† Alexander S. Kisleyou,‡ Ronald G. Harvey,‡ Thomas J. Slaga,† Rebecca J. Morris,§ and Moon-shong Tang*,† Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park-Research Division, Smithville, Texas 78957, Lankenau Medical Research Center, 100 Lancaster Avenue West of City Line, Wynnewood, Pennsylvania 19096, and Ben May Institute, University of Chicago, Chicago, Illinois 60637 Received July 2, 1996X
DNA fragments modified with chemically synthesized 7,12-dimethylbenz[a]anthracene antidiol epoxide (anti-DMBADE) are sensitive to UvrABC nuclease incision. The incisions occur mainly 7 bases 5′ and 4 bases 3′ of an anti-DMBADE-modified adenine or guanine residue, and the kinetics of incision at different sequences in a DNA fragment are the same. These results indicate that UvrABC incision on anti-DMBADE-DNA adducts is independent of DNA sequences and is quantitative, the same as on syn-DMBADE-DNA adducts. This method was used to analyze the anti-DMBADE-DNA binding spectrum in the exon 2 region of the mouse H-ras gene, and it was found that anti-DMBADE binds to the two adenine residues at codon 61 of the H-ras gene with an average affinity. Previously, we have demonstrated that synDMBADE binds strongly to the adenines at codon 61 of H-ras; these results together suggest that the oncogenic mutation in H-ras may be induced by anti- and syn-DMBADE-DNA adducts.
Introduction 7,12-Dimethylbenz[a]anthracene (DMBA)1 is a potent carcinogen (1-3). It has been shown that metabolically activated syn- and anti-dihydrodiol epoxides react with DNA at the excyclic amino moiety of adenine and guanine; this binding occurs at the benzylic position 1 of the diol epoxide, causing the epoxide ring to open to form different isomers of adducts (Figure 1) (4-10). It has been suggested that these DNA adducts trigger carcinogenesis (11, 12); however, the relative contributions of the adducts induced by these two isomeric forms of DMBADE in mutagenesis and carcinogenesis remain unclear. One distinct molecular characteristic associated with DMBA-initiated tumorigenesis is that the oncogenic activation occurs exclusively in the ras gene family and the involved mutations occur mainly in codon 61 of the ras genes (12-16). Since activated DMBA can potentially adduct the purine residues in codons 12, 13, and 61 (17) and, furthermore, any mutation in codon 12, 13, or 61 of the H-ras gene can trigger tumorigenesis (1822), one interesting and important question raised from these results is why most, if not all, DMBA-induced skin papillomas have a mutation in codon 61 but not in codon 12 or 13 (12-16). It is possible that activated DMBA diol epoxide may not form DNA adducts at codons 12 and 13 efficiently and/or the adducts formed in these sequences are repaired swiftly and efficiently. In order to unravel this important question, we have recently de* Corresponding author. † University of Texas. ‡ University of Chicago. § Lankenau Medical Research Center. X Abstract published in Advance ACS Abstracts, November 1, 1996. 1 Abbreviations: DMBA, 7,12-dimethylbenz[a]anthracene; antiDMBADE, 7,12-dimethylbenz[a]anthracene anti-diol epoxide; synDMBADE, 7,12-dimethylbenz[a]anthracene syn-diol epoxide; H-ras, Havey-ras.
S0893-228x(96)00111-7 CCC: $12.00
Figure 1. Reaction mechanism of anti- and syn-DMBADE with DNA. R represents guanine or adenine residue in DNA.
veloped a method, using the Escherichia coli nucleotide excision enzyme UvrABC nuclease, to detect syn-DMBADE-DNA adducts at the nucleotide sequence level (17). In this report, we have characterized the mode of incision of UvrABC toward DNA modified with chemically synthesized DMBA-anti-diol epoxide (anti-DMBADE). We have found that the UvrABC nucleases are able to incise anti-DMBADE-DNA adducts quantitatively and specifically. Using this method, we have determined the anti-DMBADE binding spectrum in a DNA fragment which includes exon 2 region of the mouse H-ras gene.
Materials and Methods Materials. Restriction enzymes, T4 polynucleotide kinase, and DNA polymerase I (Klenow fragment) were obtained from New England BioLabs (Beverley, MA). Calf intestinal alkaline phosphatase was purchased from Promega (Madison, WI). NACS Prepac convertible columns were supplied by Bethesda Research Labs (Gaithersburg, MD). All other chemicals and
© 1996 American Chemical Society
anti-DMBADE-DNA Binding Specificity in H-ras Gene electrophorectic materials were obtained from either Sigma (St. Louis, MO) or Bio-Rad (Hercules, CA). [R-32P]dCTP and [γ-32P]ATP (specific activity approximately 3000 Ci/mmol) were purchased from DuPont New England Nuclear (Boston, MA). anti-DMBADE Modification of DNA. The racemic antiDMBADE was synthesized according to the method reported previously (23). anti-DMBADE is extremely unstable, and it converts to the tetrahydro form in aqueous solution almost instantaneously. Different concentrations of anti-DMBADE were prepared by diluting the freshly synthesized compound in dimethyl sulfoxide. Restriction fragments isolated from the mouse H-ras gene were 32P-end-labeled as previously described (17). The 32P-labeled DNA fragments were dissolved in 90 µL of TE buffer (10 mM Tris and 1 mM EDTA, pH 7.8), mixed with 10 µL of different concentrations of freshly prepared chemicals, and incubated at 25 °C for 2 h. Unreacted anti-DMBADE was removed by repeated phenol, diethyl ether extractions and followed by ethanol precipitation in the presence of 2. 5 M ammonium acetate. The DNA pellet was washed with 70% ethanol and dried in vacuum. UvrABC Nuclease Reactions. The UvrABC nuclease reactions were carried out in a reaction mixture (25 µL) containing 50 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 10 mM MgCl2, 1 mM ATP, 100 mM KCl, 1 mM dithiothreitol, 15 nM UvrA, 15 nM UvrB, 15 nM UvrC, and substrate DNA as described previously (17). The mixture was incubated at 37 °C for 1 h, and the reactions were stopped by phenol and diethyl ether extractions and followed by ethanol precipitation in the presence of aqueous ammonium acetate (2.5 M). The precipitated DNA was then washed with 70% ethanol and dried under vacuum. DNA Sequencing, Gel Electrophoresis, and Autoradiography. The single 5′- and 3′-end-32P-labeled DNA fragments were sequenced by the method of Maxam-Gilbert (24). The 32P-labeled DNA fragments with or without UvrABC nuclease treatments were suspended in sequencing tracking dye (80% v/v deionized formamide, 0.1% xylene cyanol, and 0.1% bromophenol blue), heated at 90 °C for 4 min, and quenched in an ice bath. The samples were applied to a 0.4-0.8-mm denaturing sequencing gel consisting of 8% acrylamide and 7 M urea in TBE buffer (50 mM Tris-HCl, 50 mM borate, and 10 mM EDTA, pH 8.3). The gels were dried in a Bio-Rad gel dryer, initially exposed to a phosphor screen and then to Kodak X-Omat RP film at -70 °C for various lengths of time. The intensity of bands was determined by a PhosphorImager (Molecular Dynamics).
Results anti-DMBADE-DNA Modified DNA Fragments Are Sensitive to UvrABC Incision. In order to test whether UvrABC nucleases are able to recognize and incise anti-DMBADE-DNA adducts, DNA fragments labeled with 32P at a single 5′- or 3′-end were reacted with different concentrations of anti-DMBADE solution and then reacted with UvrABC nucleases. Figure 2 shows the results of electrophoretic separations of the UvrABC treated DNA in a denaturing polyacrylamide gel. When the anti-DMBADE modified 5′-end-32P-labeled DNA was reacted with UvrABC nucleases, the general cutting bands corresponded to Maxam and Gilbert purine reaction ladders, but were 7 nucleotides smaller (Figure 2A, lane 3). When anti-DMBADE modified 3′-32P-end-labeled DNA was used as substrate, the UvrABC reaction also generated bands which corresponded to Maxam and Gilbert purine reaction ladders, but these bands were 4 nucleotides smaller (Figure 2B, lane 2). Since UvrABC nucleases do not incise unmodified DNA (data not shown), these results suggest that UvrABC makes dual incisions 7 bases 5′ and 4 bases 3′ of anti-DMBADE modified purines. This UvrABC incision pattern induced by anti-
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1351
Figure 2. Electrophoretic separation of anti-DMBADE modified DNA fragments treated with UvrABC nucleases. (A) 5′-32Pend-labeled HinfI-NheI 194 bp fragments (0.45 pmol) of the H-ras gene containing codon 61 were modified with 10-4 mg/ mL anti-DMBADE, and the modified fragments (0.09 pmol) were reacted with (lane 3) and without (lane 4) UvrABC. (B) 3′-32P-end-labeled HinfI-NheI 194 bp fragments (0.45 pmol) of the H-ras gene were modified with anti-DMBADE (10-4 mg/ mL), and the modified fragments were reacted with (lane 2) and without (lane 1) UvrABC. Lines on the left side of the panel (A) and right side of the panel (B) represent the purine positions, and lines on the right side of the panel (A) and left side of the panel (B) represent the corresponding UvrABC incision positions. The UvrABC incision bands corresponding to codon 61 position are highlighted. Double lines represent the double UvrABC incision positions on the 5′ or 3′ side of a modified purine. G and GA are the Maxam-Gilbert sequencing reactions. (+) and (-) signs represent modified DNA fragments with UvrABC treatment and with mock treatment, respectively.
DMBADE modification is the same as we previously found by syn-DMBADE modification (17). UvrABC Nucleases Cut anti-DMBADE-DNA Adducts Quantitatively, and the Incisions Are Sequence Independent. The intensity of anti-DMBADEinduced UvrABC incision bands, shown in Figure 2, varies at different sequences, similar to syn-DMBADEinduced UvrABC incision (17); this could be due to different affinities of anti-DMBADE modification at different sequences or could be due to sequence-dependent incision by UvrABC nuclease. We previously resolved this issue by reacting syn-DMBADE modified DNA fragments with molar excessive UvrABC nucleases and subsequently analyzing incision kinetics at different sequences; we concluded that UvrABC nucleases incise syn-DMBADE-DNA adducts quantitatively and the incisions are sequence independent (17). We have undertaken the same approach for determining the nature of UvrABC incision on anti-DMBADE modified DNA fragments. Figure 3A shows the electrophoresis results of the DNA reacted with molar excessive UvrABC nucleases
1352 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Figure 3. The time course of UvrABC incision on antiDMBADE modified DNA fragments. 5′-32P-end-labeled HinfINheI 194 bp fragments of the H-ras gene (0.45 pmol) were modified with anti-DMBADE (1.0 × 10-4 mg/mL). The modified DNA fragments (0.09 pmol) were then incubated with UvrABC nucleases (lanes 4-7) for different periods of time (5, 10, 40, and 80 min), and the resultant DNAs were separated in a 8% polyacrylamide denaturing gel as described in Figure 2A. Lane 3 is the modified DNA without UvrABC nuclease treatment. The trinucleotide nucleotide sequences (with * representing the anti-DMBADE modification sites) which correspond to the UvrABC incision bands are depicted on the right side of the panel. The intensity of each UvrABC incision band was quantified in a PhosphorImage. (A) Autoradiograph, and (B) the kinetics of UvrABC incisions at different sequences in the antiDMBADE modified DNA fragments. The intensity of UvrABC cutting bands at different sequences in the anti-DMBADE modified DNA fragments with different incubation time was normalized to the highest intensity of UvrABC cutting bands among the same sequences. For clarity, only two sequence points are presented in (B); the rest of sequence points (total of 8) are within the length of the bar. The solid lines represent the average value of the different sequences. Since the amount DNA loaded in the gel from the sample of 20 min incubation was 60% of the other sample, for visional clarity, the cutting bands from this sample were not included in panel A; however, their relative intensities were calculated and included in panel B.
Chen et al.
for different time periods. From visual inspection, it appears that the intensity of the UvrABC incision band at different sequences is a function of incubation time. A total of 8 bands in a well-separated region were chosen for quantification, and the results in Figure 3B show that UvrABC incises DNA adducts formed in these sequences with identical kinetics and that the incision plateaus after 40 min of incubation. Since the molar ratio of UvrABC/DNA is 4.3 in these reaction conditions, and, furthermore, because the UvrABC incision is an irreversible reaction, these results strongly suggest that DNA sequence does not play a significant role in determining the efficiency of UvrABC incision and that the degree of UvrABC incision should be proportional to the extent of anti-DMBADE-DNA binding. Binding Spectrum of anti-DMBADE-DNA Adducts in the Exon 2 Region of the Mouse H-ras Gene. Since our results show that under our standard reaction conditions the intensity of UvrABC incision at different sequences should represent the sequence preference of anti-DMBADE-DNA binding, the UvrABC incision method can, therefore, be used to determine the sequence selectivity of anti-DMBADE-DNA. To determine the anti-DMBADE-DNA binding spectrum in the exon 2 region of the mouse H-ras gene, the intensity of each UvrABC incision band in Figure 2A,B was quantified by a PhosphoImager. Figure 4 shows the calculated relative intensity at different sequences obtained from either the 5′-end (upper panel) or the 3′-end (lower panel) 32 P-labeled DNA fragments. Bands in the high molecular region were poorly separated and therefore were not quantified; the overlapping region represents sequences which were well separated in both the 5′- and 3′-endlabeled fragments. Results in Figure 4 demonstrate the following: (1) the relative intensity of UvrABC cutting at the 5′-side is comparable with the intensity of the 3′side cutting; these results suggest that either 5′- or 3′end-labeled fragments can be used for determining the anti-DMBADE-DNA binding spectrum; and (2) in comparison with other sequence, the adenines in codon 61 do not bind to anti-DMBADE strongly; however, the second adenine has a significantly higher affinity for antiDMBADE binding than the first adenine. We have analyzed the extent of guanine and adenine adductions in the exon 2 of H-ras gene based on UvrABC incision analysis. For all the cutting bands, the average values of relative intensity were calculated in Table 1. The results show that anti-DMBADE binds significantly higher with guanines than with adenines with a ratio of 1.42; these results are identical with HPLC analysis results (4). In contrast, syn-DMBADE binds adenines preferentially, with a guanine/adenine adduction ratio of 0.8 (17).
Discussion Chemically synthesized anti-DMBADE is extremely unstable; the compound converts to an inactive tetrahydro form rapidly, even under rigid anaerobic conditions. Harvey et al. (23), however, have previously confirmed that the compound synthesized by the current protocol is indeed anti-DMBADE by spectroscopic analysis. Because of the unstable nature of this compound, we were not successful in determining the effect on UvrABC incision by the different concentrations of anti-DMBADE used for DNA modification. Nonetheless, we have found that DNA fragments modified with 10-4 mg/mL antiDMBADE under our conditions are sensitive to UvrABC
anti-DMBADE-DNA Binding Specificity in H-ras Gene
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1353
Figure 4. The DNA binding spectrum of anti-DMBADE in the NheI-HinfI fragment of coding strand of exon 2 in the mouse H-ras gene. The DNA fragments were 5′-end (upper panel) or 3′-end (lower panel) labeled with 32P, modified with anti-DMBADE, reacted with UvrABC nucleases, and then separated by gel electrophoresis, as described in Figure 2. The intensity of UvrABC incision bands in well-separated regions was quantified in a PhosphorImager. The extent of anti-DMBADE-DNA binding is represented by the relative intensity (RI) of UvrABC incision bands. The RI was calculated based on RI ) Ii/Imax, where Ii is the intensity of each UvrABC incision band and Imax is the UvrABC incision band with the highest intensity in an autoradiograph. The dotted line represents the average intensity of the UvrABC incision bands. Table 1. Binding Ratio of Adenine and Guanine at Exon 2 Region of H-ras by syn- and anti-DMBADE syn-DMBADEa RIb
RIb
anti-DMBADE RIb
av of A site
av of G site
ratio of G/A
av of A site
av RIb of G site
ratio of G/A
0.283 (36)
0.225 (35)
0.8
0.330 (26)
0.468 (29)
1.42
a
The values were calculated from data presented in ref 15. b RI is relative intensity calculated from binding spectrum. Average (av) RI is calculated from dividing total RI by total binding sites. The number of analyzed sites is shown in parentheses.
incision and DNA modified with 10-3 and 10-2 mg/mL anti-DMBADE is resistant to UvrABC incision (data not shown). We have previously observed similar results: that is, DNA modified with high concentrations of anthramycin, CC-1065, mitomycin, and syn-DMBADE is a poor substrate for UvrABC nuclease (17, 25-28). This is likely due to the interaction of two or more DNA adducts from the same DNA molecule or different molecules and results in forming tangled DNA molecules which are no longer recognizable by UvrABC nucleases. Our results have clearly shown that UvrABC nuclease incises anti-DMBADE-DNA adducts in the same fashion as it incises other DNA damage, such as pyrimidine dimers and aminofluorene-, (acetylamino)fluorene-, and benzo[a]pyrene diol epoxide-DNA adducts (25, 27, 29, 30); that is, it incises 7 bases 5′ and 4 bases 3′ to an antiDMBADE-purine adduct. We have also shown that this cutting is both specific and quantitative. We previously demonstrated that UvrABC is also able to incise synDMBADE-DNA adducts specifically and quantitatively (17); together, these results suggest that the UvrABC incision method can be used for quantifying DMBAinduced DNA damage in cells. Our results show that the binding affinities for both syn- and anti-DMBADE are generally similar, but there are some notable differences between the two; for example, at sequences -CAG-, -CAC-, -CAA-, and -CAT- synDMBADE binds more strongly than anti-DMBADE, and the former binds to the adenines at codon 61 more strongly than the latter (17). In contrast, at sequences -TGG-, -GGA-, -AGA-, -AGG-, -GGT-, and -AGA- anti-
DMBADE binds more strongly than syn-DMBADE. These results are consistent with our statistical data shown in Table 1, which indicate that syn-DMBADE binds adenines better than guanines and that anti-DMBADE binds guanines better than adenines. Although these results are consistent with those detected by HPLC (4), the chemical nature which causes this differential binding for these two bases in DNA is unclear. Cheng et al. (31) have shown that in cellular systems synthetic racemic syn-DMBADE reacts predominately with adenine, while racemic anti-DMBADE reacts with both guanine and adenine. It is conceivable that in vivo the DMBADE-DNA binding spectrum may be different from the in vitro binding spectrum due to not only the composition of racemic activated DMBA and their reactivity but also the effects of chromatin structure, nuclear protein association, and gene activity, and it is likely that these factors may also affect the repair of the DMBADEDNA adducts. All of these factors may also contribute to the tissue specificity of DMBA-induced oncogenic mutation of a particular member of ras gene family. Our finding that UvrABC nucleases are able to incise DMBADE-DNA adducts specifically and quantitatively provides us with a useful tool to determine the binding in vivo and to investigate the effects of these factors.
Acknowledgment. We thank Ms. A. Pao and Mr. Y. Zheng for preparing Uvr proteins and Ms. Y.-Y. Tang for critical review of the manuscript. This work was supported by Grants ES03124 (M.-s.T.), CA45293 (R.J.M.), and CA57596 (T.J.S.) from the United States Public Health Service, Grant 3955 (M.-s.T.) from the Council for Tobacco ResearchsUSA, Inc., and Grant CN22 (R.G.H.) from the American Chemical Society. J.X.C. is also supported by NIH Postdoctoral Training Grant CA09480.
References (1) Slaga, T. J., Gleaso, G. H., DiGiovanni, J., Sukumaran, K. B., and Harvey, R. G. (1979) Potent tumor initiating activity of the 3,4-dihydrodiol of 7,12-dimethylbenz[a]anthracene in mouse skin. Cancer Res. 39, 1934-1936.
1354 Chem. Res. Toxicol., Vol. 9, No. 8, 1996 (2) Bigger, C. A. H., Sawicki, J. T., Blake, D. M., Raymond, L. G., and Dipple, A. (1983) Products of binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse skin. Cancer Res. 43, 5647-5651. (3) Manam, S., Storer, R. D., Prahalada, S., Leander, K. R., Kraynak, A. R., Edwith, B. J., Van Zwieten, M. J., Bradley, M. O., and Nichols, W. W. (1992) Activation of the Ha-, Ki-, and N-ras gene in chemically induced liver tumors from CD-1 mice. Cancer Res. 52, 3347-3362. (4) DiGiovanni, J., Fisher E. P., and Sawyer, T. W. (1986) Kinetics of formation and disappearance of 7,12-dimethylbenz[a]anthracene: DNA adducts in mouse epidermis. Cancer Res. 46, 4400-4405. (5) Dipple, A., Pigott, M. A., Bigger, C. A. H., and Blake, D. M. (1984) Dimethylbenz[a]anthracene-DNA binding in mouse skin; response of different mouse strains and effects of various modifiers of carcinogenesis. Carcinogenesis 5, 1087-1090. (6) Bigger, C. A. H., Tomaszewski, J. E., and Dipple, A. (1980) Variation in route of microsomal activation of 7,12-dimethylbenz[a]anthracene with substrate concentration. Carcinogenesis 1, 15-20. (7) Bigger, C. A. H., Tomaszewski, J. E., Dipple, A., and Lake, R. S. (1980) Limitations of metabolic activation systems used with in vitro tests for carcinogens. Science (Washington, DC) 209, 503505. (8) Cooper, C. S., Ribeiro, O., Hewer, A., Walsh, C., Grover, P. L., and Sims, P. (1980) Additional evidence for the involvement of the 3,4-diol 1,2-oxides in metabolic activation of 7,12-dimethylbenz[a]anthracene in mouse skin. Chem.-Biol. Interact. 29, 357367. (9) Sawick, J. T., Moschel, R. C., and Dipple, A. (1983) Involvement of both syn- and anti-dihydrodiol-epoxides in the binding of 7,12-dimethylbenz[a]anthracene to DNA in mouse embryo cell cultures. Cancer Res. 43, 3212-3218. (10) Vericat, J. A., Cheng, S. C., and Dipple, A. (1989) Absolute stereochemistry of the major 7,12-dimethylbenz[a]anthraceneDNA adducts formed in mouse. Carcinogenesis 10, 567-570. (11) Balmain, A., Ramsden, M., Bowden, G.T., and Smith, J. (1984) Activation of the mouse cellular Harvey-ras gene in chemically induced being skin papillomas. Nature 307, 658-660. (12) Quintanilla, M., Brown, K., Ramsden, M., and Balmain, A. (1986) Carcinogen-specific mutation and amplification of Ha-ras during mouse skin carcinogenesis. Nature 322, 78-80. (13) Quintanilla, M., Haddow, S., Jonas, D., Jaffe, D., Bowden, G.-T., and Balmain, A. (1991) Comparison of ras activation during epidermal carcinogenesis in vitro and in vivo. Carcinogenesis 12, 1875-1881. (14) Bizub, D., Wood, A. W., and Skalka, A. M. (1986) Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycylic aromatic hydrocarbons. Proc. Natl. Acad. Sci. U.S.A. 83, 60486052. (15) Robles, A. I., Gimenez-Conti, I. B., Roop, D., Slaga, T. J., and Conti, C. J. (1993) Low frequency of codon 61 Ha-ras mutations and lack of keratin 13 expression in 7,12-dimethylbenz[a]anthracene-induced hamster skin tumors. Mol. Carcinog. 7, 9498. (16) Gimenez-Conti, I. B., Sharon, A. B., Stockman, S. L., Conti, C. J., and Slaga, T. J. (1992) Activating mutation of the Ha-ras gene in chemically induced tumors of the hamster cheek pouch. Mol. Carcinog. 5, 259-263.
Chen et al. (17) Chen, J. X., Pao, A., Zheng, Y., Ye, X., Kisleyou, A., Morris, R., Slaga, T. J., Harvey, R., and Tang, M.-s. (1996) Sequence preference of 7,12-dimethylbenz[a]anthracene-syn-diol epoxideDNA binding in mouse H-ras gene detected by UvrABC nucleases. Biochemistry 35, 9594-9602. (18) Balmain, A., and Brown, K. (1988) Oncogene activation in chemical carcinogenesis. Adv. Cancer Res. 51, 147-182. (19) Sukumar, S. (1990) An experimental analysis of cancer: Role of ras oncogenes in multistep carcinogenesis. Cancer Cell 2, 199204. (20) Barbacid, M. (1987) Ras genes. Annu. Rev. Biochem. 56, 779827. (21) Bos, J. L. (1989) Ras Oncogenes in human cancer: A review. Cancer Res. 49, 4682-4689. (22) Manam, S., Store, R. D., Prahalada, S., Leander, K. R., Kraynak, A. R., Hammermeister, C. L., Joslyn, D. J., Ledwith, B. J., Zweiten, M. J., Bradley, M. O., and Nichols, W. W. (1992) Activation of the Ki-ras gene in spontaneous and chemically induced lung tumors in CD-mice. Mol. Carcinog. 6, 68-75. (23) Lee, H., and Harvey, R. G. (1986) Synthesis of the active diol epoxide metabolite of the potent carcinogenic hydrocarbon 7,12dimethylbenz[a]anthrene. J. Org. Chem. 51, 3502-3507. (24) Maxam, A. M., and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499. (25) Tang, M.-s., Pierce, J. R., Doisy, R. P., Nazimiec, M. E., and MacLeod, M. C. (1992) Differences and similarities in the repair of two benzo[a]pyrene diol epoxide isomers induced DNA adducts by uvrA, uvrB, and uvrC gene products. Biochemistry 31, 8429. (26) Nazimiec, M. E., Grossman, L., and Tang, M.-s. (1992) A comparison of the rate of reaction and function of UVRB- and UVRAB-mediated anthramycin-N2-guanine-DNA repair. J. Biol. Chem. 267, 24716-24724. (27) Pierce, J. R., Case, R., and Tang, M.-s. (1989) Recognition and repair of 2-aminoflurene- and 2-(acetylamino)fluorene DNA adducts by UVRABC nuclease. Biochemistry 28, 5821-5826. (28) Tang, M.-s., Lee, C.-S., Doisy, R., Ross, L., Needham-VanDevanter, D. R., and Hurley, L. H. (1988) Recognition and repair of the CC-1065-(N3-adenine)-DNA adduct by the UvrABC nucleases. Biochemistry 27, 893-901. (29) Seeberg, E., and Steinum, A. L., Nordenskjold, M., Soderhall, S., and Jernstrom, B. (1983) Strand-break formation in DNA modified by benzo[alpha]pyrene diolepoxide. Quantitative cleavage by Escherichia coli UvrABC endonuclease. Mutat. Res. 112, 139145. (30) Sancar, A., and Rupp, W. D. (1983) A novel repair enzyme: UvrABC excision nuclease of E. coli cuts a DNA strand on both sides of the damaged region. Cell 33, 249-60. (31) Cheng, S. C., Prakash, A. S., Pigott, M. A., Hilton, B. D., Lee, H., Harvey, R. G., and Dipple, A. (1988) A metabolite of the carcinogen 7,12-dimethylbenz[a]anthracene that reacts predominantly with adenine residues in DNA. Carcinogenesis 9, 17211723.
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