Quantitative and Qualitative Analysis of DNA Methylation at N3

Chem. Res. Toxicol. 1998, 11, 1481-1486

1481

Quantitative and Qualitative Analysis of DNA Methylation at N3-Adenine by N-Methyl-N-nitrosourea Jack D. Kelly,† Dharini Shah,†,‡ Fa-Xian Chen,† Richard Wurdeman,† and Barry Gold*,†,‡ Eppley Institute for Research in Cancer and Allied Diseases and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68105 Received July 24, 1998

The sequence-specific alkylation of DNA by N-methyl-N-nitrosourea (MNU) has been demonstrated for the minor groove N3-methyladenine (N3-MeAde) adduct using neutral thermal hydrolysis and polyacrylamide sequencing gels. The ratio of relative yields of N7- and N3MeAde and N7-methylguanine (N7-MeGua) is approximately 0.03:0.15:1.00, respectively, on the basis of the gel data, and these values are comparable to relative yields determined by bulk digestion of MNU-methylated DNA when HPLC was used to analyze the individual adducts. In contrast to the methylation at N7-guanine (N7-Gua) by MNU, alkylation at Ade shows minimal sequence selectivity. Similar to the methylation at N7-Gua, formation of N3MeAde by MNU is inhibited by 50-200 mM concentrations of NaCl and DNA binding cations, including distamycin and spermine. However, N3-MeAde formation at Ade residues within methidiumpropyl-EDTA-Fe(II) footprinted distamycin DNA affinity binding regions is selectively inhibited at low concentrations of distamycin relative to Ade sites outside of ligand binding regions, and N7-Gua within or outside the distamycin binding regions. HPLC analysis shows that distamycin also quantitatively inhibits the production of N3-methylguanine when calf thymus DNA is treated with MNU or methyl methanesulfonate. The specific inhibitory effect of distamycin, which binds in the minor groove at Ade/Thy-rich sequences, provides additional evidence that the predominant DNA lesion detected at Ade by sequencing gel analysis involves minor groove N3-MeAde modifications.

Introduction The sequence-dependent modification of DNA by carcinogens and related antineoplastic agents may be considerably important in understanding at a molecular level how these agents exert their biological activity, i.e., cytotoxicity and mutagenicity. At the same time, this information can also provide a sensitive probe of sequencedependent changes in DNA structure and properties. We have previously explored the effect of sequence on the alkylation of DNA in the major groove at N7-Gua by N-methyl-N-nitrosourea (MNU)1 (1-3). MNU shows a modest sequence-dependent reaction with DNA (1-3), and the selectivity is similar to that observed with other alkylating agents that react via charged intermediates, e.g., chloroethylnitrosoureas (4), alkyl triazenes (5), and nitrogen mustards (6-8). The combined results show that there is a clear relationship between sequence and electrophilic substitution at this major groove site. The yield of O6-methylguanine (O6-MeGua), another major groove adduct, has also been reported to show a sequencedependent behavior similar to that observed for N7-Gua when DNA is treated with MNU (9-11). * To whom correspondence should be addressed. Phone: (402) 5595148. Fax: (402) 559-4651. E-mail: [email protected]. † Eppley Institute for Research in Cancer and Allied Diseases. ‡ Department of Pharmaceutical Sciences. 1 Abbreviations: bp, base pair; DMS, dimethyl sulfate; MMS, methyl methanesulfonate; MNU, N-methyl-N-nitrosourea; MPE, methidiumpropyl-EDTA-Fe(II); N3-MeAde, N3-methyladenine; N3-MeGua, N3methylguanine; N7-MeAde, N7-methyladenine; N7-MeGua, N7-methylguanine.

While the major groove N7-MeGua product is quantitatively the predominate DNA lesion generated by MNU, it has limited biological activity (12). There is overwhelming evidence for the role of O6-MeGua in the mutagenicity and toxicity of DNA methylating agents even though it is a relatively minor product [10% relative to N7-MeGua (13, 14)]. Another adduct formed in low yield is N3-MeAde. Despite the yield, this lesion appears to play a dominant role in the cytotoxicity of alkylating agents (15, 16). Previously, the effect of sequence on Ade methylation has only briefly been addressed (17). Herein, we detail a more complete characterization of the methylation of DNA in the minor groove by MNU.

Experimental Procedures Caution: MNU and MMS should be considered toxic and potential human carcinogens and should be handled accordingly. MNU, methyl methanesulfonate (MMS), and distamycin A were purchased from Aldrich Chemical Co. (Milwaukee, WI), and fresh solutions were prepared immediately prior to their use. All other chemical reagents were of the highest available purity. Restriction enzymes, alkaline phosphatase, and T4 kinase were purchased from Bethesda Research Laboratories (Bethesda, MD). Reaction of Alkylating Agents with Calf Thymus DNA. Calf thymus DNA (500-600 µM) was treated at room temperature with MNU in 100 mM Tris-HCl buffer (pH 7.8) or MMS in 10 mM Tris-HCl buffer (pH 7.8) (incubation time and concentration of methylating agent and distamycin described in Table 1). At the end of the incubation, the DNA was precipitated, washed with cold EtOH, and dissolved in 10 mM

10.1021/tx9801763 CCC: $15.00 © 1998 American Chemical Society Published on Web 11/07/1998

1482 Chem. Res. Toxicol., Vol. 11, No. 12, 1998

Kelly et al.

Table 1. Adduct Yields from the Reaction of Calf Thymus DNA with MMS and MNU adduct yielda compound

N7-MeGua

N3-MeGua

N3-MeAde/N7-MeGua

N3-MeAde/N3-MeGua

5.0 mM 5.0 mM and distamycinc MNUe 10.0 mM 10.0 mM and distamycin

0.68 ( 0.08 ndd

3.47 ( 0.34 3.54 ( 0.24

0.08 ( 0.02 nd

0.17 -

8.5 -

1.75 ( 0.11 nd

18.84 ( 2.27 11.43 ( 1.90

0.12 ( 0.02 nd

0.09 -

14.6 -

MMSb

d

adduct ratio

N3-MeAde

a Picomoles of adduct per microgram of DNA. b Incubation time of 24 h at ambient temperature. c Distamycin concentration of 100 µM. nd means not detected. e Incubation time of 4 h at ambient temperature.

Tris-HCl buffer (pH 7.8). The DNA solution was heated at 90 °C for 30 min to preferentially release N3- and N7-alkylpurines from DNA (18). The reaction mixture was then left overnight at 0 °C and the remaining oligomeric DNA precipitated by the addition of 10% (v/v) ice-cold 1.0 N HCl. The supernatant containing the N-alkylated purines was removed from the oligomeric DNA by centrifugation and dried in vacuo. HPLC Analysis of Adducts. The N-alkylated purines were dissolved in 10 mM Tris buffer (pH 7.8) and separated on a Varian HPLC system: YMC ODS-18 AQ column (4.6 mm); temperature, 40 °C; and solvent, 0.1 M sodium acetate (pH 5.2) containing 5% MeOH. The N3-MeAde adduct was quantitated with a Varian UV photodiode array detector. The N7-MeGua and N3-MeGua adducts were analyzed using an ESA Coulochem II detector: guard cell voltage, 850 mV; analytical cell voltage, 800 mV; and range, 100 nA. Preparation of 5′-32P-End-Labeled DNA Restriction Fragments. The 85 and 576 bp 5′-32P-end-labeled DNA fragments were isolated from a 3220 bp DNA clone containing the promotor region of the coat protein gene of the canine parvovirus (19) as previously described (2) using standard methods (20). Reactions of MNU with 32P-End-Labeled DNA Restriction Fragments. The restriction fragment (80000-100000 cpm) and sonicated calf thymus DNA (final concentration of 83 µM) were dissolved in 10 mM Tris-HCl buffer (pH 7.8) containing the desired concentration of NaCl or cationic DNA affinity binder. DNA solutions were incubated with freshly prepared solutions of MNU at 37 °C for 4 h. The reactions were terminated by cooling and precipitation of the DNA with NaOAc and EtOH. The DNA was washed with cold 70% EtOH and dried in vacuo. Generation and Analysis of DNA Strand Breaks. Strand breaks in the reacted DNA were generated by one of the following methods. (1) For neutral thermal hydrolysis, methylated DNA was heated at 90 °C for 15 min to depurinate and/or depyrimidinate any thermally labile adducts, precipitated with NaOAc and EtOH, washed with cold EtOH, and then treated with hot piperidine to convert the abasic sites into single-strand breaks (21). (2) For Maxam-Gilbert Gua-specific reaction, alkylated DNA was treated as above except the precipitated DNA was not heated at neutral pH, but directly treated with 1 M piperidine at 90 °C for 20 min to preferentially convert N7alkylpurines into single-strand breaks (20). (3) For mild acid workup, alkylated DNA was treated with ammonium formate (pH 2.0) for 30 min at 0 °C to selectively depurinate N3-MeAde, N7-MeAde, and N7-MeGua and then precipitated with NaOAc and EtOH, dried, and heated with 1 M piperidine (20). In all cases, the piperidine was removed in vacuo. In control experiments, no alkylating agent was added. The standard Gua and Gua+Ade reaction lanes (20) were included as sequence markers. The DNA samples were denatured at 90 °C for 1.5 min and then cooled in ice/water prior to electrophoresis at 65 W on a 12% polyacrylamide gel containing 7.8 M urea and Tris-EDTA borate buffer (pH 8.3). The gel was exposed to Kodak X-OMAT AR film at -70 °C and the resulting autoradiogram analyzed using a Shimadzu CS-9000 scanning densitometer. DNA Footprinting. Using 5′-32P-end-labeled restriction fragments and sonicated calf thymus DNA (83 µM final concentration), the affinity binding regions of distamycin were

Figure 1. Base sequences of the resolved portion of the 85 (Figure 2a) and 576 (Figure 2b) bp restriction fragments. The underlined bases are protected from MPE-induced cleavage by 10 µM distamycin. footprinted with MPE as previously described (22). The dried DNA was then suspended in loading buffer, denatured, and run on polyacrylamide gels as described above.

Results The analysis of N3-MeAde, N3-MeGua, and N7-MeGua adducts was carried out by treating calf thymus DNA with the different alkylating agents at concentrations and incubation times that roughly afforded similar level of adducts. Some incubations were carried out in the presence of distamycin, which is known to equilibrium bind to Ade/Thy-rich regions (23-26). The adducts were released from DNA using neutral thermal hydrolysis and quantitated using HPLC with UV and electrochemical detection. The EC detection was required to quantitate N3-MeGua, which is a minor adduct. The absolute and relative yields of the adducts are shown in Table 1. In the absence of added distamycin, the relative yields of the three adducts from MNU are similar to what has been previously reported. However, distamycin quantitatively inhibits the MNU- and MMS-mediated formation of the two minor groove products, i.e., N3-MeAde and N3MeGua. There is an approximately 40% decrease in the yield of N7-MeGua from MNU in the presence of distamycin, while no decrease is observed with MMS (Table 1). Using 32P-labeled restriction fragments (see Figure 1 for sequences) and taking into account the relative thermal and acid lability of N3-MeAde, N3-MeGua, N7MeAde, and N7-MeGua (21), we could visualize the location and intensity of these adducts (Figure 2) using high-resolution polyacrylamide sequencing gels and autoradiography (20). It is well-known that MMS does not

N3-Methyladenine Sequence Specificity

a

Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1483

b

Figure 2. Autoradiograms of a 12% polyacrylamide gel used to map the methylation pattern of MNU in the 85 and 576 bp restriction fragments employing neutral thermal hydrolysis (90 °C for 15 min) followed by piperidine treatment (unless stated otherwise). The distamycin footprinted sites are indicated by a bar in the sequence legend: (a) 85 bp fragment and (b) 576 bp fragment.

react with DNA with any sequence dependency (1, 20). This is one of the reasons why alkyl sulfonate esters are used in the chemical sequencing of DNA (20). In addition, cations have no significant effect on the methylation of DNA by MMS (1, 20). Analysis of the incubations of the 85 bp fragment with MNU demonstrates a clear dose-response relationship for cleavages at both Gua and Ade sites (Figure 2a) within the range of concentrations studied. The average densitometrically determined ratio of cleavage at Ade to Gua is 0.16 compared to 0.09 with the HPLC analysis (Table 1). The use of selective depurination of N3-MeAde and N7-MeGua at pH 2.0 gave similar results (Figure 2a). The bands that correspond to Ade cleavage sites are not observed when the MNU-treated DNA is directly treated with hot piperidine (Figure 2a), a procedure that selectively cleaves at N7-modified purines.

The MNU-mediated DNA cleavage at Ade is inhibited by increasing salt concentrations (Figure 2a, lanes i and j) and, in this regard, is very similar to what is observed at Gua (1, 2). The cationic DNA affinity binder spermine (Figure 2a) also causes a similar sequence-independent reduction at both Ade and Gua cleavage sites. The low concentrations of ethidium bromide (1-10 µM) have no effect on DNA methylation (Figure 2a), but in previous studies using higher concentrations, we have observed an inhibitory effect of ethidium bromide on the yield of N7-MeGua (1). There is no strong sequence specificity for the formation of the N3-MeAde adducts; all Ade residues are cleaved approximately to the same extent, with the exception being Ade207 in the 85 bp fragment. This Ade site also shows hypersensitivity to dimethyl sulfate using Maxam-Gilbert Gua chemistry (Figure 2a). However,

1484 Chem. Res. Toxicol., Vol. 11, No. 12, 1998

the carbethoxylation of Ade207 at the N7-position by diethylpyrocarbonate (26) shows no enhancement compared to that at the other Ade sites (data not shown). It is most likely that this band is a combination of N3MeAde and some incomplete piperidine cleavage products arising from methylation at Gua209 (20). For the remaining Ade cleavage sites, the range of the difference in the relative intensities is approximately 2-fold between the most intense (Ade229) and the least intense (Ade211) bands (Figure 2a). The effect of distamycin on Ade methylation by MNU was also assessed (Figure 2). The affinity binding recognition sites of distamycin were footprinted using MPE on the 85 and 576 bp fragments, and the sequences protected from cleavage are shown in Figure 1, and in the sequence legend in Figure 2. There is a pronounced reduction in the band intensity at Ade residues within the ligand’s binding sites as compared to the intensity of those outside of the binding regions (Figure 2). The bands assigned to Ade225, Ade211, Ade207, and Ade205 in the 85 bp fragment are not in distamycin binding domains. Distamycin (10 µM) reduces the intensity of these bands by 45-50%, while the intensity of the Ade sites in affinity binding domains is reduced by 73-96% (Figure 2a). The selective effect of distamycin on Ade positions in the 576 bp fragment is also evident at Ade (Figure 2b). Ade269 and Ade270 in distamycin binding regions are strongly inhibited, while Ade273 and Ade276, which are not in affinity binding regions, are weakly affected. At this low concentration of distamycin, the intensities of Gua cleavage sites are not altered (Figure 2).

Discussion The methylation of DNA by the carcinogen MNU involves its hydrolysis to methanediazotic acid, which in turn ionizes to methanediazonium ion (1, 2, 27-31). The cationic diazonium ion is generally accepted as the species responsible for covalent adduction. The involvement of a cationic alkylating intermediate is consistent with the global inhibition of DNA methylation by added cations (Figure 2a), and the regiospecific inhibition by tethered ammonium ions (32, 33). On the basis of multiple literature reports, methylation at N7- and N3Gua, and N3- and N7-Ade by MNU in calf thymus or salmon sperm DNA, as determined by DNA digestion and HPLC quantitation, accounts for approximately 66, 0.6, 8, and 2%, respectively, of the total DNA modifications (13, 14). The data in Table 1, which does not include data from the quantitation of N7-MeAde, and the sequencing gel results are consistent with these data. The densitometric quantitation of the gel data shows an average ratio for the cleavage at Ade to Gua of 0.16. This is the similar to the value of 0.09 derived from the digestion of mammalian DNA coupled with HPLC analysis of the adducts (Table 1) and literature values that range from 0.15 to 0.17 (13, 14). The correspondence between the gel-derived data and that from DNA digestion with HPLC analysis is probably within experimental error. It should be pointed out that the cleavage bands at Ade in Figure 2 reflect the sum of two types of modifications: N3-MeAde and N7-MeAde (13, 14). Both Ade lesions are subject to thermal or weak acid-catalyzed depurination. The N7-MeAde adduct, similar to N7MeGua, can undergo base-catalyzed imidazole ring open-

Kelly et al.

ing, but the ring-opened adduct is only “eliminated” by nucleophilic bases, e.g., piperidine (34, 35). The literature value for the ratio of N7-MeAde to N7-MeGua is 0.03 DNA (13). The observed decrease in DNA cleavage at Ade in the presence of cations, i.e., NaCl, spermine, and distamycin (Figure 2), is similar to that seen at Gua sites. It is assumed that the concentration-dependent neutralization of the electrostatic attraction between the positively charged methanediazonium ion and the negatively charged DNA by inorganic salts and the cationic DNA affinity binders is responsible for this global decrease in the extent of formation of adducts at Ade. This inhibitory effect, which is sequence-independent, has been previously addressed in relation to the formation of methylated DNA adducts from MNU (1, 2, 9, 27, 36-41). In addition to the electrostatic influence on DNA methylation, it was anticipated that distamycin would selectively inhibit adduction at N3-Ade atoms within the antibiotic’s affinity binding domains. Distamycin, which is a monocation that binds to the minor groove of DNA at Ade/Thy-rich regions by a combination of H-bonds and van der Waals contacts (25), should physically and electrostatically block the access of the methanediazonium ion to the minor groove of DNA at its affinity binding site. The data (Figure 2) support this prediction and, along with the absence of Ade bands with MaxamGilbert chemistry, provide evidence that the predominant Ade lesion visualized by autoradiography is a consequence of the minor groove N3-MeAde adduct. The presence of the distamycin ligand has significantly less effect on the cleavage intensities of Ade residues that are outside of the distamycin’s affinity binding sites. Because we have no sequence data for the trace N3-MeGua adduct, it is not possible to comment on how its sequencedependent formation is inhibited by distamycin. It is possible that the distamycin transiently binds in the minor groove to regions that contain Gua‚Cyt base pairs, albeit at a much lower affinity, or that a modest decrease in the yield of the N3-MeGua adduct drops the level below the limit of detection. It should be noted that in previous studies the inhibitory effect of distamycin was reported at N7-Gua, O6-Gua, and N3-Ade using an HPLC analysis, although specific values for the extent of inhibition at the different sites were not presented (38). In our HPLC and gel studies, using low concentrations of distamycin, we observe selective inhibition of minor groove adducts. This inhibition is striking and could provide a method for preparing DNA that is virtually devoid of minor groove-methylated lesions. Whether this effect can be translated to the in vivo methylation of DNA is under study. Sequence Specificity. The sequence specificity for methylation at N7-Gua by MNU has been reported, and within (Gua)3 runs, the central base can be methylated as much as 8 times more than the flanking bases (1, 2). In contrast, this work indicates that the effect of base sequence on band intensities at Ade is less pronounced. The resolved regions of the DNA restriction fragments studied contain CAT, TAA, AAC, AAT, TAT, GAT, AAA, AAG, CAA, GAA, TAC, and CAC sequences (Figure 1). With the exception of a single Ade, the difference between Ade cleavage intensities is approximately 2-fold; the intensity of the most intense cleavage site in the 85 bp fragment (CA207C) is about twice the average intensity using neutral thermal or mild acid hydrolysis. The more

N3-Methyladenine Sequence Specificity

intense cleavage at CA207C, which is also seen with dimethyl sulfate using Maxam-Gilbert Gua conditions (Figure 2a), may result from methylation at N7-Ade since this chemistry is specific for N7-alkylpurine adducts. However, the fragmentation pattern induced by diethylpyrocarbonate (data not shown), which modifies N7Ade, does not show enhanced reactivity of Ade207 under Gua cleavage reaction conditions. Therefore, it does not appear that Ade207 is in a noncanonical region of DNA (42). The most logical explanation for the aberrant intensity at Ade207 is that the band is a composite of N3MeAde cleavage and heterogeneous termini generated by piperidine cleavage at N7-MeGua223 (20). The general similarity in the alkylation at all Ade’s implies that the accessibility and nucleophilicity of the Ade residues are nearly equivalent. In fact, it has been suggested that the general inaccessibility of N3-Ade in duplex DNA may be limiting in N3-Ade alkylation, and mask any electrostatic effect that neighboring bases would have on reactivity (39). Breslauer et al. (43) have shown that the enthalpic and entropic components of the free energy for the affinity binding of cationic drugs to DNA dramatically vary with DNA sequence. Thus, the affinity binding of distamycin to poly[d(Ade-Thy)]-poly[d(Ade-Thy)] is enthalpy-driven, while the binding to homopolymers poly(dAde)-poly(dThy) is entropy-driven. The overall free energies of binding are comparable. Similar results were seen for the binding of the related dication netropsin (43). The change in driving forces for binding, without a concomitant change in the total binding free energy, is explained by different degrees of hydration of the two DNA duplexes. If an electrostatic interaction between the charged methanediazonium ion and DNA is important in the alkylation process, and this certainly appears to be the case, then sequence-specific hydration of the minor groove could result in a masking of any intrinsic differences in Ade reactivity.

Acknowledgment. This work was supported by U.S. Public Health Service Research Grant CA29088 and Cancer Center Support Grant CA36727 awarded by the National Cancer Institute and by American Cancer Society Center Grant ACS SIG-16.

References (1) Wurdeman, R. L., and Gold, B. (1988) The effect of DNA sequence, ionic strength, and cationic DNA affinity binders on the methylation of DNA by N-methyl-N-nitrosourea. Chem. Res. Toxicol. 1, 146-147. (2) Wurdeman, R. L., Church, K. M., and Gold, B. (1989) DNA methylation by N-methyl-N-nitrosourea, N-methyl-N′-nitro-Nnitrosoguanidine, N-nitroso(1-acetoxyethyl)methylamine and diazomethane. The mechanism for the formation of N7-methylguanine in sequence-characterized 5′-[32P]-end-labeled DNA. J. Am. Chem. Soc. 111, 6408-6412. (3) Wurdeman, R. L., Douskey, M. C., and Gold, B. (1993) DNA methylation by N-methyl-N-nitrosourea: methylation pattern changes in single- and double-stranded DNA, and in DNA with mismatched or bulged guanines. Nucleic Acids Res. 21, 49754980. (4) 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. (5) Hartley, J. A., Mattes, W. B., Vaughan, K., and Gibson, N. W. (1988) DNA sequence specificity of guanine N7-alkylations for a series of structurally related triazines. Carcinogenesis 9, 669674. (6) Mattes, W. B., Hartley, J. A., and Kohn, K. W. (1986) DNA sequence selectivity of guanine-N7 alkylation by nitrogen mustards. Nucleic Acids Res. 14, 2971-2987.

Chem. Res. Toxicol., Vol. 11, No. 12, 1998 1485 (7) Kohn, K. W., Hartley, J. A., and Mattes, W. B. (1987) Mechanisms of DNA sequence selective alkylation of guanine-N7 positions by nitrogen mustards. Nucleic Acids Res. 15, 10531-10549. (8) Hartley, J. A., Forrow, S. M., and Souhami, R. L. (1990) Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards. Biochemistry 29, 2985-2991. (9) 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. (10) Richardson, F. C., Boucheron, J. A., Skopek, T. R., and Swenberg, J. A. (1989) Formation of O6-methylguanosine at specific sites in a synthetic oligonucleotide designed to resemble a known mutagenic hot spot. J. Biol. Chem. 264, 838-841. (11) Dolan, M. E., Oplinger, M., and Pegg, A. E. (1988) Sequence specificity of guanine alkylation and repair. Carcinogenesis 9, 2139-2143. (12) Lawley, P. D. (1984) Carcinogenesis by alkylating agents. In Chemical Carcinogens (Searle, C. E., Ed.) Vol. 1, pp 325-484, American Chemical Society, Washington, DC. (13) 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, 595-605. (14) 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, 393403. (15) Engelward, B., Dreslin, A., Christensen, J., Huszar, D., Kurahara, C., and Samson, L. (1997) Repair deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation induced chromosome damage and cell killing. EMBO J. 15, 945-952. (16) Engelward, B. P., Allan, J. M., Dreslin, A. J., Kelly, J. D., Gold, B., and Samson, L. D. (1998) 3-Methyladenine DNA lesions induce chromosome aberrations, cell cycle delay, apoptosis and p53. J. Biol. Chem. 273, 5412-5418. (17) Church, K. M., Wurdeman, R. L., Zhang, Y., Chen, F.-X., and Gold, B. (1990) N-(2-Chloroethyl)-N-nitrosoureas covalently bound to nonionic and monocationic lexitropsin dipeptides. Synthesis, DNA affinity binding characteristics, and reactions with 32P-end-labeled DNA. Biochemistry 29, 6827-6838. (18) Bianchini, F., Montesano, R., Shuker, D. E. G., Cuzick, J., and Wild, C. P. (1993) Quantitation of 7-methyldeoxyguanosine using immunoaffinity purification and HPLC with electrochemical detection. Carcinogenesis 14, 1677-1682. (19) Rhode, S. L., III (1985) Nucleotide sequence of the coat protein gene of canine parvovirus. J. Virol. 54, 630-633. (20) Maxam, A. M., and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65, 499-560. (21) Lawley, P. D., and Brookes, P. (1963) Further studies on the alkylation of nucleic acids and their constituent nucleotides. Biochem. J. 89, 127-138. (22) Van Dyke, M. W., Hertzberg, R. P., and Dervan, P. B. (1982) Map of distamycin, netropsin, and actinomycin binding sites on heterogeneous DNA: DNA cleavage-inhibition patterns with methidiumpropyl-EDTA. Proc. Natl. Acad. Sci. U.S.A. 79, 54705474. (23) Zimmer, C. (1975) Effects of antibiotics netropsin and distamycin A on the structure and function of nucleic acids. Prog. Nucleic Acid Res. Mol. Biol. 15, 285-318. (24) Taylor, J. S., Schultz, P. G., and Dervan, P. B. (1984) DNA affinity cleaving. Sequence specific cleavage of DNA by distamycin-EDTAFe(II) and EDTA-distamycin-Fe(II). Tetrahedron 40, 457-465. (25) Kopka, M. L., Yoon, C., Goodsell, D., Pjura, P., and Dickerson, R. E. (1985) The molecular origin of DNA-drug specificity in netropsin and distamycin. Proc. Natl. Acad. Sci. U.S.A. 82, 1376-1380. (26) Leonard, N. J., McDonald, J. J., Henderson, R. E. L., and Reichmann, M. E. (1971) Reaction of diethyl pyrocarbonate with nucleic acid components. Adenosine. Biochemistry 10, 3335-3342. (27) Kriek, E., and Emmelot, P. (1964) Methylation of deoxyribonucleic acid by diazomethane. Biochim. Biophys. Acta 91, 59-66. (28) White, E. H., and Woodcock, D. J. (1968) Cleavage of the carbonnitrogen bond. In The Chemistry of the Amino Group (Patai, S., Ed.) pp 440-483, Wiley-Interscience, New York. (29) 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 vivo with methyl methanesulphonate and N,N-dimethylnitrosamine. Biochem. J. 129, 519-528.

1486 Chem. Res. Toxicol., Vol. 11, No. 12, 1998 (30) Magee, P. N., Nicoll, J. W., Pegg, A. E., and Swann, P. F. (1975) Alkylating intermediates in nitrosamine metabolism. J. Chem. Soc. Trans. 1975, 62-65. (31) Gold, B., Deshpande, A., Linder, W., and Hines, L. (1984) Reactions of alkanediazotic acids at near neutral and basic pH in [18O]H2O. J. Am. Chem. Soc. 106, 2072-2077. (32) Liang, G., Encell, L., Nelson, M. G., Switzer, C., and Gold, B. (1995) The role of electrostatics in the sequence selective reaction of charged alkylating agents with DNA. J. Am. Chem. Soc. 117, 10135-10136. (33) Dande, P., Liang, G., Chen, F.-X., Roberts, C., Switzer, C., and Gold, B. (1997) The regioselective effect of zwitterionic DNA substitutions on DNA alkylation: Evidence for a strong side-chain orientational preference. Biochemistry 36, 6024-6032. (34) Maxam, A. M., and Gilbert, W. (1977) A new method for sequencing DNA. Proc. Natl. Acad. Sci. U.S.A. 74, 560-564. (35) 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. (36) McCalla, D. R. (1968) Reaction of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitroso-p-toluenesulfonamide with DNA in vitro. Biochim. Biophys. Acta 155, 114-120. (37) Jensen, D. E., and Reed, D. J. (1978) Reaction of DNA with alkylating agents. Quantitation of alkylation by ethylnitrosourea

Kelly et al.

(38)

(39) (40) (41) (42) (43)

of oxygen and nitrogen sites on poly[dA-dT] including phosphotriester formation. Biochemistry 17, 5098-5107. Rajalakshmi, S., Rao, P. M., and Sarma, D. S. R. (1978) Studies on carcinogen chromatin-DNA interaction: inhibition of Nmethyl-N-nitrosourea-induced methylation of chromatin-DNA by spermidine and distamycin A. Biochemistry 17, 4515-4518. Pullman, A., and Pullman, B. (1981) Molecular electrostatic potential of the nucleic acids. Q. Rev. Biophys. 14, 289-380. 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. 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. Peattie, D. A., and Gilbert, W. (1980) Chemical probes for higherorder structure in RNA. Proc. Natl. Acad. Sci. U.S.A. 77, 46794682. Breslauer, K. J., Remeta, D. P., Chou, W.-Y., Ferrante, R., Curry, J., Zaunczkowski, D., Synder, J. G., and Marky, L. A. (1987) Enthalpy-entropy compensations in drug-DNA binding studies. Proc. Natl. Acad. Sci. U.S.A. 84, 8922-8926.

TX9801763