146
Chen. Res. Toxicol. 1988, I , 146-147
The Effect of DNA Sequence, Ionic Strength, and Cationic DNA Affinity Binders on the Methylation of DNA by N-Methyl-N-Nitrosourea
Sic DNA alkylation by N-alkyl-N-nitrosoureas (1) is generally accepted to be responsible for their mutagenic, carcinogenic, and antineoplastic activities (2). The exact nature of the ultimate alkylating intermediate is still controversial, with a variety of species having been nominated (Figure 1)(3-5).The sequence specificity for DNA alkylation by simple N-alkyl-N-nitrosoureas has not been reported, although such information is basic in understanding the specific point mutations induced by these compounds in oncogene targets (6). These two points are addressed by using N-methyl-N-nitrosourea (MNU) methylation of a 576 base-pair (bp) 32P-end-labeled DNA restriction fragment (7)and high-resolution polyacrylamide sequencing gels. This method provides information on the formation of N-methylguanine (N7-MeG), by the generation of single-strand breaks upon exposure to piperidine (8). As shown in the autoradiogram (Figure 2) the formation of N7-MeG from 50 mM dimethyl sulfate (DMS) is qualitatively and quantitatively the same in the absence of salt or affinity binders (lane B), in the presence of 50, 100, and 200 mM NaCl (lanes C-E), 100 pM distamycin A (lane F), 10 pM spermine (lane G), or 10 pM ethidium bromide (lane H). In contrast, methylation by 1 mM MNU (lane K) is dose-dependently inhibited by NaCl (lanes M-P), distamycin A (lanes Q-S), spermine (lanes T-V), and ethidium bromide (lanes W-Y). The N7-MeG pattern caused by MNU is not qualitatively altered by salt or the cationic DNA affinity binders. Another obvious difference between DMS and MNU methylation is the pattern of intensities a t the oligo d(G) runs located a t G263-G266, Gm1-Gm and GD3-GD5(see Figure 2 for sequence). Finally, with MNU there is a definite preference for N7-MeG formation at these dG runs. I t is not likely that the change in salt concentration is qualitatively or quantitatively affecting the ultimate electrophilic intermediate because (a) the hydrolysis of N-alkyl-N-nitrosoureas is subject to specific base catalysis (9, IO),which is insensitive to ionic strength (9), and (b) both sN2 and s N 1 pathways (Figure 1) will be enhanced a t high ionic strength (11). The possibility that the addition of salt causes a conformational change that decreases the accessibility of nucleophilic DNA sites is not attractive since alkylation a t G3, 06G, G7, A3, 02Tand the phosphate backbone oxygens are all similarly inhibited (12-15). Also, the methylation of single-stranded RNA is diminished at high ionic strength (12). The data are consistent with a strong electrostatic attraction (at low salt) between the polyanionic DNA backbone (16) and a positively charged alkylating intermediate (14). Accordingly, the sN2 alkylation of DNA by dimethyl sulfate (Figure 2) is inhibited by neither salt nor the cationic affinity binders. The screening of the electrostatic DNA potential by salt or the strong cationic binders, as previously proposed (14), appears responsible for the observed decrease in alkylation (17,18). It is also possible that the inhibition of N7-MeG formation a t high concentrations of NaCl (lanes M-P) could result from a competition of C1- with DNA for the methylating intermediate. The distamycin A and spermine-mediated inhibition of DNA methylation by MNU has previously been observed (19). Both drugs inhibited methylation a t 06and N7-G, but were ineffective at inhibiting the adduction of 5'-deoxyguanylic acid. A t low concentrations these drugs are known to bind specifically to A-T rich regions of DNA (20,21), but their inhibition
DNFl a1 kylation
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--
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N=N
'
\OH
R
I
DNFl alkylation ( sN2
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Figure 1. Pathways for the hydrolysis of N-alkyl-N-nitrosoureas to yield SN2 (alkanediazoticacid) and S N 1 (alkanediazonium ion) DNA alkylating intermediates.
.- ' . -
1 "
-,.".
.'.
.... . .
.
- -
. .. ..
Figure 2. Autoradiogram of 12% polyacrylamide gel (65 W)used to map 1 M piperidine-induced (heated at 90 "C for 20 min) single-strand breaks caused by N7-guanine adduction by MNU and DMS. 32P-end-labeled restriction fragment (7)was incubated with MNU or DMS at 37 "C for 2 h in Tris-HCl buffer (pH 8.0): lane A, control; lanes B-H, 50 mmM DMS (Maxam-Gilbert G); lanes C-E, 50,100, and 200 mM NaCl, respectively; lane F, 100 pM distamycin A; lane G, 10 pM spermine; lane H, 10 pM ethidium bromide; lane I, Maxam-Gilbert G+A; lanes J, 2 mM MNU; lanes K and M-Y, 1 mM MNU; lane L, 0.5 mM MNU; lanes M-P, 50,100,150, and 200 mM NaCl; lanes Q-S,50,100, and 200 pM distamycin A; lanes T-V, 1,10,and 100pM spermine; lanes W-Y,1,10, and 100pM ethidium bromide. Approximately the first 100 bp from the 5'-terminus (bp 11239) of the fragment are resolved in the autoradiogram.
of MNU methylation shows no sequence-dependence at any concentration (lanes Q-V). The preference of MNU for oligo d(G) sequences is reminiscent of the DNA alkylation pattern induced by N-(2-chloroalkyl)-N-nitrosoureas(22). The high electrostatic potential predicted for such sequences may account for this interesting selectivity (16). Finally, the mechanism 0 1988 American Chemical Society
Communications
Chem. Res. Toxicol., Vol. 1, No. 3, 1988 147
recently proposed by Buckley to account for high MNUmediated methylation of d(G)-d(C)is not consistent with the results in Figure 2, since the predicted selectivity for the 3’-G in d(G),, runs is not observed (5). Acknowledgment. We thank Dr. Solon Rhode for the Parvovirus plasmid and his helpful comments. This work was supported by NIH Grant CA29088 and Laboratory Cancer Research Grant CA36727 awarded by the National Cancer Institute. Registry No. MNU, 684-93-5; DMS, 77-78-1; N7-G, 578-76-7.
References (1) For review, see: Beranek, D. T., Weis, C. C., and Swenson, D. H. (1980) “A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography”. Carcinogenesis 1,595606. Den Engelse, L., Menkveld, G . J., De Brij, R.-J., and Tates, A. D. (1986) “Formation and stability of alkylated pyrimidines and purines (including imidazole ring-opened 7-alkylguanine) and alkylphosphotriesters in liver DNA of adult rats treated with ethylnitrosourea or dimethylnitrosamine”. Carcinogenesis 7, 393-403. (2) For review, see: Genotonicity of N-Nitroso Compounds (Rao, T. K., Lijinsky, W., and Epler, J. L., Eds.) Plenum, New York, (1984). Preussmann, R., and Stewart, B. W. (1984) “N-Nitroso carcinogens”. In Chemical Carcinogens (Searle, C. E., Ed.) ACS Monograph 182, pp 643-828, American Chemical Society, Washington, DC. (3) An SN2attack by nucleophile on an alkanediazotic acid has been proposed by: Park, K. K., Archer, M. C., and Wishnok, J. S. (1980) “Alkylation of nucleic acids by N-nitrosodi-n-propylamine: evidence that carbonium ions are not significantly involved”. Chem.-Biol. Interact. 29, 139-144. Lown, J. W., Chauhan, S. M. S., Koganty, R. R., and Sapse, A.-M. (1984) “Alkyldinitrogen species implicated in the carcinogenic, mutagenic, and anticancer activities of N-nitroso compounds: characterization by 15N NMR of 16N-enriched compounds and analysis of DNA base situselectivity by ab initio calculations”. J . Am. Chem. SOC.106, 6401-6408. (4) An SN1pathway from the alkanediazonium ion or from a nitrogen-separated ion pair has been proposed by: Kriek, E., and Emmelot, P. (1964) “Methylation of deoxyribonucleic acid by diazomethane”. Biochim. Biophys. Acta 91,59-66. O’Connor, P. J., Capps, M. J., Craig, A. W., Lawley, P. D., and Shah, S. A. (1972) “Differences in the patterns of methylation in rat liver ribosomal ribonucleic acid after reaction in uiuo with methyl methanesulphonate and N,N-dimethylnitrosamine”. Biochem. J. 129,519-528. Magee, P. N., Nicoll, J. W., Pegg, A. E., and Swann, P. F. (1975) ”Alkylating intermediates in nitrosamine metabolism”. J . Chem. SOC.,Perkin Trans. 1 62-65. White, E. H., and Woodcock, D. J. (1968) “Cleavage of the carbon-nitrogen bond”. In The Chemistry of the Amino Group (Patai, S., Ed.) pp 409-497, Wiley-Interscience, New York. Gold, B., Deshpande, A., Linder, W., and Hines, L. (1984) “Reactions of alkane diazotic acids a t near neutral and basic pH in [180]H,0”. J . Am. Chem. SOC. 106, 2072-2077. (5) Recently a mechanism involving an intramolecular attack by a nucleophilic DNA site on a tetrahedral complex, formed by a general base-catalyzed reaction, has been proposed by: Buckley, N. (1987) “A regioselective mechanism for mutagenesis and oncogenesis caused by alkylnitrosourea sequence-specific DNA 109,7918-7920. This mechanism alkylation“. J . Am. Chem. SOC. requires that a G that is 3’ to another G will be a preferred alkylation site. (6) Zarbyl, H., Sukumar, S., Arthur, A. V., Martin-Zanca, D., and Barbacid, M. (1985) “Direct mutagenesis of Ha-ras-1 oncogenes
by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats”. Nature (London) 315, 382-385. (7) The fragment was isolated from a 3220 bp DNA clone of the coat protein gene of the canine parvovirus [Rhode, S. L., 111. (1985) “Nucleotide sequence of the coat protein gene of canine parvovirus”. J . Virol. 54,6304331 by NCOI digestion, treatment with calf intestine alkaline phosphatase, phosphorylation with T4 kinase in the presence of [ T - ~ ~ P ] Aand T P Hind111 restriction (: ). Two 32P-end-labeledfragments (576 and 85 bp) were isolated by gel purification. (8) Maxam, A. M., and Gilbert, W. (1980) “Sequencing end-labeled DNA with base-specific chemical cleavages”. Methods Enzymol. 65, 499-560. Mattes, W. B., Hartley, J. A., and Kohn, K. W. (1986) “Mechanism of DNA strand breakage by piperidine at sites of N7-alkylguanines”. Biochim. Biophys. Acta 868, 71-76. (9) Garrett, E. R., Goto, S., and Stubbins, J. F. (1965) “Kinetics of solvolysis of various N-alkyl-N-nitrosoureas in neutral and alkaline solutions”. J . Pharm. Sci. 54, 119-123. (10) Synder, J. K., and Stock, L. M. (1980) “Reactions of alkylnitrosoureas in aqueous solution”. J . Org. Chem. 45, 1990-1999. (11) High ionic strength will accelerate the SN2pathway because neutral reactants generate a charged product (OH-). The SN1 mechanism involves generation of an alkanediazonium ion in a charged transition state and will also be enhanced at high salt. (12) Kriek, E., and Emmelot, P. (1963) “Methylation and breakdown of microsomal and soluble ribonucleic acid from rat liver by diazomethane”. Biochemistry 2, 733-740. (13) McCalla, D. R. (1968) “Reaction of N-methyl-N’-nitro-Nnitrosoguanidine and N-methyl-N-nitroso-p-toluenesulfonamide with DNA in uitro”. Biochim. Biophys. Acta 155, 114-120. (14) Jensen, D. E., and Reed, D. J. (1978) “Reaction of DNA with alkylating agents. Quantitation of alkylation by ethylnitrosourea of oxygen and nitrogen sites on poly[dA-dT] including phosphotriester formation”. Biochemistry 17, 5098-5107. (15) Briscoe, W. T., and Cotter, L.-E. (1985) “DNA sequence has an effect on the extent and kinds of alkylation of DNA by a potent carcinogen”. Chem.-Biol. Interact. 56, 321-331. (16) For review, see: Pullman, A., and Pullman, B. (1981) “Molecular electrostatic potential of the nucleic acids”. Q. Reu. Biophys. 14, 289-380. (17) Zakrzewska, K., and Pullman, B. (1983) “A theoretical evaluation of the effect of netropsin binding on the reactivity of DNA towards alkylating agents”. Nucleic Acids Res. 11, 8841-8845. (18) Zakrzewski, K., and Pullman, B. (1985) “The effect of spermine binding on the reactivity of DNA towards carcinogenic alkylating agents”. J . Biomol. Struct. Dyn. 3, 437-444. (19) Rajalakshmi, S., Rao, P. M., and Sarma, D. S. R. (1978) “Studies on carcinogen chromatin-DNA interaction: inhibition of N-methyl-N-nitrosourea-induced methylation of chromatinDNA by spermidine and distamycin A”. Biochemistry 17, 4515-4518. (20) Schultz, P. G., Taylor, J. S., and Dervan, P. B. (1982) “Design and synthesis of a sequence-specific DNA cleaving molecule. (Distamycin-EDTA)iron(II)”. J. Am. Chem. SOC. 104, 6861-6863. (21) Drew, H. R., and Dickerson, R. E. (1981) “Structure of B-DNA dodecamer 111. Geometry of hydration”. J . Mol. Biol. 151, 535-536. (22) Hartley, J. A., Gibson, N. W., Kohn, K. W., and Mattes, W. B. (1986) “DNA sequence selectivity of guanine-N7 alkylation by three antitumor chloroethylating agents”. Cancer Res. 46, 1943-1947.
Richard L. Wurdeman, Barry Gold*
Eppley Institute for Research i n Cancer and Allied Diseases University of Nebraska Medical Center Omaha, Nebraska 68105 Received January 27, 1988
