Carcinogenesisvol.il no.6 pp.947-951, 1990
Tlhe reaction off TV-metlhiyll-A'-mtrosoiarea wfflh BNA in IB and mom-IB
Jamie R.Milligan, Sandra M.Skotnicki and Michael C.Archer1 Department of Medical Biophysics, University of Toronto, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9 'To whom correspondence should be addressed
Introduction We have recently shown that the pattern of DNA methylation is identical for three different /V-nitroso compounds—Nnitroso(acetoxymethyl)methylamine, A'-nitroso(acetoxybenzy 1) methylamine and N-methyl-A'-nitrosourea (MNU*) (1). This result suggests that methylation by these compounds occurs via a common reactive intermediate such as the methyl diazonium ion. We observed, however, that significantly less 7-methylguanine is formed in poly(dG-dC) • poly(dG-dC) than in poly(dG)-poly(dC), calf thymus DNA or supercoiled plasmid DNA. These results are in general agreement with the similar observations of Briscoe and Cotter for MNU (2,3). We also showed that there is some sequence specificity in the interaction of DNA with the methylating agents (1). The maximum difference in reactivity of specific guanines is ~ 5-fold, which might explain the differences we observed in reactivity of the various DNA substrates. However, the effect of 20% DMSO that we used to aid the solubility of the carcinogens may have prompted the B to Z transition in poly(dG-dC) • poly(dG-dC) (4,5), which may have affected the reactions. The conditions used by Briscoe and Cotter (15.5% ethanol) may also have favored this transition (4,5). Secondary structure is known to influence the reaction with DNA of several small molecules including diethylpyrocarbonate (6,7), bromoacetaldehyde (8), chloroacetaldehyde (9,10), hydroxylamine (11), osmium tetroxide (11), permanganate (7) and ozone (12). Similar behavior has also been observed in the reaction of some well-studied chemical carcinogens with DNA. For example N-hydroxy-2-aminofluorene and iV-acetoxy-2-acetylaminofluorine (N-acetoxy-AAP), react with B DNA but not with Z DNA (8,9). N-Acetoxy-AAF, however, can react at specific DNA sites with unpaired bases in supercoiled plasmid DNA that 'Abbreviations: MNU, A'-methyl-iV-nitrosourea; N-acetoxy-AAF, /V-acetoxy2-acetylaminofluorine; TE, 10 mM Tris/1 mM EDTA, pH 7.4; TBE, 0.089 M Tris-borate/0.025 M EDTA, pH 8.3. © Oxford University Press
Materials and methods Plasmids Plasmid samples were generously provided by David E.Pulleyblank (University of Toronto). The plasmids contained the following inserts ligated into the Smal site of either the vector pDPL6 (18): pDHgl6, d(GQ, 2 d(GQ 1 2 (19); pAT34/6, d(AT) l7 -d(AT)| 7 (20); or the vector p913 (21): pGA34, d(GA) n -d(TQ, 7 (22). They were prepared from Escherichia coli K12 HB101 by conventional methods (23). Preparation of topological isomers Topological isomers of the plasmids were prepared by topoisomerase I catalyzed relaxation in the presence of 0, 10 or 20 nM ethidium (24). Ten rrucrograms of plasmid DNA were reacted for 4 h at 37°C with 6 U topoisomerase I in a total volume of 100 /J under conditions recommended by the manufacturer (BRL, Gaithersburg, MD). Four hundred microlitres of TE (10 mM Tris/1 mM EDTA, pH 7.4) were then added to the reaction and the enzyme and ethidium were removed by extraction with TE-saturated phenol (2 x 500 pX), a 1:1 mixture of TE-saturated phenol and a 4% (v/v) solution of isoamyl alcohol in chloroform (2 x 500 pi), and 4% isoamyl alcohol in chloroform (2 x 500 jil). After extensive dialysis, the DNA was finally dissolved in TE. The distribution of topoisomers was determined by two-dimensional agarose gel (1 %) electrophoresis (25). The first dimension was run in TBE (0.089 M Tris-borate/0.025 M EDTA, pH 8.3) and the second dimension in TBE containing 0.5 /ig/ml chloroquine phosphate. The superhelical densities (a) of the three plasmids used were 0, -0.029 and -0.058 at 0, 10 and 20 jdvl ethidium respectively. Plasmids treated with topoisomerase I in the absence of ethidium were fully relaxed, while those in 20 pM ethidium were strongly negatively supercoiled. DNA modification reactions Five rrucrograms of plasmid DNA were dissolved in 50 /J TE and reacted with 0.03 mM MNU at room temperature overnight. Reaction conditions for diethylpyrocarbonate, chloroacetaldehyde and dimethylsulfate were as previously described (see refs 15, 9 and 21 respectively). pGA34 was preincubated in 20 mM acetate, pH 5, for 60 min before the reactants were added. DNA sequencing After incubation of the chemically modified plasmids with restriction endonudeases (Hindm and Pstl for pDHgl6; Sail and Pstl for PAT34/6; Xbal and BstEU for pGA34) under conditions recommended by the manufacturer (BRL), the fragments containing the inserts were isolated by preparative agarose gel electrophoresis, electroelution and hydroxyapathe chromatography (26), followed by extensive diafiltration with TE. The restriction fragments from pDHgl6, pAT34/6 and pGA34 were labeled at the 3' ends of the HindlH, Sail or flnEII sites with [a-^PJdATP, [a-^PJdGTP or [a-^PldGTP respectively using the Klenow fragment of DNA polymerasc I. The radioactive DNA was recovered by gel filtration (Sephadex G50) followed by ethanol precipitation after addition of non-radioactive carrier DNA. After lypophilization, the DNA samples were treated with 100 pi 1 M piperidine at 90°C for 30 min, then run on a pre-electrophoresed 20% polyacrylamide—8 M urea gel as described by Maxam and Gilbert (27). The intensities of individual bands on one of the sequencing gel autOTadiographs were
947
Downloaded from http://carcin.oxfordjournals.org/ at Monash University on June 22, 2015
Reaction of W-methyl-iV-nitrosourea (MNU) with DNA in B and non-B conformations was investigated using plasmids containing inserts in which the non-B topological isomers could readily be prepared by topoisomerase I catalyzed relaxation in the presence of ethidium. DNA sequences in the Z, cruciform and H conformations were shown to be methylated by MNU at the N7 position of guanines or the N3 position of adenines in a manner that was indistinguishable from the reaction of MNU with the same sequences in the B conformation. Electronic factors rather than steric factors, therefore, appear to dominate methylation of DNA by MNU.
assume non-B structures (9). There is also evidence that carcinogen-modified DNA can assume the Z conformation more readily than unmodified DNA (15). In order to distinguish between the possible effects of sequence specificity and conformation in the reaction of carcinogens that are simple methy lating agents with DNA, we have examined the reaction of MNU with DNA that is known to be in the B or Z conformation (16). Moreover, we have extended these studies to include DNA in the cruciform conformation (16), and a structure in which dA:dT Watson-Crick base pairs alternate with protonated Hoogsteen (syn) dG:dC pairs (16), a form that is also known as H-DNA (17).
J.R.Milligan el al. quantified densitometrically with a Beckman DU50 spectrophotometer (Fullerton, CA) and gel scanning accessory.
2 4 6 8 10 12 1 3 5 7 9 II 13 i i i
*
i i
i
i t i
i
i i i
T
1 5
— 0 -
0
Fig. 1. Agarose gel electrophorcctk analysis of the reactivity of the d(GC) l2 insert of pG16 in the B and Z conformations. Topologica] isomers of pDHgl6 were prepared by topoisomerase I catalyzed relaxation in the presence of 0 /1M (lanes 3, 6 and 9), 10 jtM (lanes 4, 7 and 10) or 20 *»M (lanes S, 8 and 11) ethidium. Isomers were reacted with chloroacetaldehyde (lanes 3 - 5 ) , diethylpyrocarbonate (lanes 6 - 8 ) or MNU (lanes 9-11). The W/ndlll—Pstl restriction fragment was then end labeled, cleaved with piperidine and electrophoresed as described in Materials and methods. Lanes 1, 2, 12 and 13 are the Maxam and Gilbert G, G + A, C + T and C calibrations respectively
948
10
20
30
Gel Length (cm)
Fig. 2. Densitometnc scans of (a) lane 11 (Z-DNA + MNU) and (b) lane 9 (B-DNA + MNU) from Figure 1.
Downloaded from http://carcin.oxfordjournals.org/ at Monash University on June 22, 2015
Results To induce the B to Z, cruciform, or Hoogsteen base pair conformational changes within the plasmic inserts, we utilized the topoisomerase I catalyzed relaxation in the presence of ethidium (24). Characterization of topoisomers by two-dimensional agarose gel electrophoresis established that 20 fiM ethidium was necessary to ensure that the inserts in each case were fully in their non-B conformations. After reaction with MNU, a fragment containing the insert was isolated by restriction endonuclease digestion. Sites of formation of 7-methylguanine or 3-methyladenine were determined by breaking the DNA at these residues by treatment with piperidine and sequencing by the conventional Maxam and Gilbert technique (27). Reactivity of guanines or adenines in the inserts in their non-B conformations was in each case compared directly to the same sequences in the B conformation (prepared in the absence of ethidium). As positive controls, we used diethylpyrocarbonate, chloroacetaldehyde or dimethylsulfate, three chemicals that are known to react anomalously with DNA in non-B conformations (6-10,21,28-30). Figure 1 shows the sequencing gels of the ~ 1 kb HindEl-Pstl fragments of pDHgl6 containing the d(GC)| 2 insert that can
assume the Z conformation. Chloroacetaldehyde is known to react extensively with cytosines at B —Z junctions (9). This hyperreactivity is seen clearly in lane 5 (20 /*M ethidium), but is absent when the insert is in the B conformation (lane 3, no ethidium). Diethylpyrocarbonate has been shown to react extensively with guanines throughout regions of Z-DNA (6,7). This may be seen clearly in lane 8 (20 fiM ethidium) in which the guanines in the insert are much more reactive than the same sequence in lanes 6 and 7 (0 and 10 /tM ethidium respectively). Lanes 9-11 show the reactivity of guanines with MNU. Since it is difficult to discern differences in these lanes by eye, particularly since the DNA loadings in each lane are not exactly equivalent, we examined two of the lanes by scanning densitometry. Figure 2a shows the scan of lane 11 in which the DNA was in the Z form (20 yM ethidium), and Figure 2b shows the scan of lane 9 in which the DNA was in the B form (no ethidium). First, it is clear from Figure 2b that all guanines in B-DNA are not equally reactive with MNU. Indeed, the peak at ~ 5 cm of a guanine flanked by other guanines is ~ 5 times more reactive than guanines within the insert which are flanked by cytosines. This sequence selectivity of guanine methylation in B-DNA agrees with our previous observations (1). Second, the guanines within the insert ( — 8—22 cm) have the same reactivity with MNU when the insert is in the B or Z conformation. Thus, the guanine peaks within the insert are approximately the same areas in Figure 2a and b when they have normalized to the peak area of a guanine outside the insert (e.g. peak at 25 cm). It is clear from the sequencing gel that several of the guanine bands in the calibration lanes 12 and 13 appear as doublets, as does the first major guanine peak above the insert in lanes 9 - 1 1 . The reason for these doublets is not clear, although it is not related to the chemical treatments since the DNA in lanes 9—11 and 12 and 13 were treated with different reagents (MNU and hydrazine respectively.) The doublets may be due to some double-
The reaction of /V-methyl-A'-nltrosourea with DNA in B and non-B conformations
2 4 6 8 10 12 14 13 5 7 9 1113 15 n i i i i i i i ii i i i i
•I III:
with guanines in the 5' half of the H-DNA region may be seen by comparing lane 3 (no ethidium) with either lane 4 (10 /tM ethidium) or lane 5 (20 /iM ethidium). The effect of supercoiling is more pronounced on the reaction of diethylpyrocarbonate with adenines (cf. lane 6 with lanes 7 and 8). However, increasing supercoiling has no discernible influence on the reactivity of MNU with guanines throughout the d(GA),7 insert (cf. lane 9 with lanes 10 and 11). Discussion
Our results demonstrate that secondary conformation plays little or no role in determining the reactivity of the N7 position of guanines or the N3 or N7 positions of adenines in DNA towards MNU. In view of the small steric demand of the methyl diazonium ion, and the greater accessibility of the N7 of guanine in Z-DNA relative to B-DNA, electronic factors appear to dominate the methylation reaction. It is clear that the diminished yield of 7-methylguanine in poly(dG-dC)-poly(dG-dC) compared to other DNA substrates that we examined in our earlier work (1) is due to the low reactivity of the N7 position of guanine when it is flanked by cytosine residues in DNA in either the B or Z conformation. In our earlier work, we observed that in contrast to the N7 position, the O 6 position of guanine had similar reactivity in poly(dG-dC)-poly(dG-dC) as in other DNA substrates (1). Therefore, the electronic factors which determine the extent of methylation at N7 by the methyldiazonium ion do not appear to affect reaction at O6. However, an elaboration of factors that affect the reactivity of O6 must await the development of methods to sequence C^-methylguanine residues.
2 I
Fig. 3. Agarose gel electrophoretic analysis of the reactivity of the d(AT)|7 insert of pAT34/6 in the B and cruciform conformations. Topological isomers of pAT34/6 were prepared by topoisomerase I catalyzed relaxation in the presence of 0 /*M (lanes 3, 7 and 11), 10 nM (lanes 4, 8 and 12) or 20 tM (lanes 5, 9 and 13) ethidium. Lanes 6, 10 and 14 show supercoiled DNA as isolated from bacteria. Isomers were reacted with chloroacetaldehyde (lanes 3 - 6 ) , diethylpyrocarbonate (lanes 7-10) or MNU (lanes 11-14). The SaR-Pst\ restriction fragment was then end labeled, cleaved with piperidine and electrophoresed as described in Materials and methods. Lanes 1, 2, and 15 are the Maxam and Gilbert G, G + A, and C + T calibrations respectively.
4 3
6 5
8 7
10 9
12 II
13
Fig. 4. Agarose gel electrophoretic analysis of the reactivity of the d(GA), 7 d(TC)| 7 insert of pGA34 in the B and H conformations Topological isomers of pGA34 were prepared by topoisomerase I catalyzed relaxation in the presence of 0 jtM (lanes 3, 6 and 9), 10 yM (lanes 4, 7 and 10) or 20 fiM (lanes 5, 8 and 11) ethidium. Isomers were reacted with dimethylsulfate (lanes 3 - 5 ) , diethylpyrocarbonate (lanes 6—8) or MNU (lanes 9-11). The Xba\-BstEU restriction fragment was then end labeled, cleaved with piperidine, and electrophoresed as described in Materials and methods. Lanes 1, 2, 12 and 13 are the Maxam and Gilbert G, G+A, C + T and C calibrations respectively.
949
Downloaded from http://carcin.oxfordjournals.org/ at Monash University on June 22, 2015
strandedness within the fragments caused by the high melting point of the d(GC)|2 insert. Figure 3 shows the sequencing gel of the ~ 1 kb SaK—Pstl fragment of pAT34/6 containing the d(AT)| 7 insert. Chloroacetaldehyde and diethylpyrocarbonate are known to show enhanced reactivity at adenines in single-stranded regions of cruciform loops (28,29). By comparing lanes 3 and 7 (no ethidium) with lanes 5 and 9 (20 /iM ethidium) or lanes 6 and 10 (unrelaxed DNA as isolated from the bacteria), it is clear that the adenines at the center of the insert that are single stranded are indeed hyper-reactive. In contrast, MNU reacts identically with adenines in the same sequence in both B and cruciform conformations, with no preference for single-stranded regions (cf. lane 11 with lanes 12-14). [Differences between lanes 11-14 are not considered to be significant. Adenines do not react extensively with MNU (31), hence produce faint bands. Small differences in loading would explain the darker bands in lane 11 compared to lanes 13 and 14.] The sequencing gel of the ~ 80 bp Xbal -BstEH fragment of pGA34 is shown in Figure 4. Both dimethylsulfate (21) and diethylpyrocarbonate (30) have been shown previously to react differentially with purines in the 3' and 5' halves of d(GA)n • d(TC)n inserts in supercoiled plasmids under acidic conditions (H-DNA). This differentia] reactivity of dimethylsulfate
J.R.MWigan et al.
Since non-B DNA structures exist in vivo and may play an important role in various genetic processes, our results showing that simple carcinogenic methylating agents typified by MNU do not show diminished reactivity with sequences in non-B conformations compared to the same sequences in the B form, may have important implications for chemical carcinogenesis. For example, carcinogen lesions in non-B DNA may be repaired differently from the same lesions in B-DNA. It is already known that E.coli formamidopyrimidine-DNA glycosylase does not remove the ring-opened form of 7-methylguanine from Z-DNA (39). Similarly, Cr-methylguanine-DNA methyltransferase from £. coli does not repair C^methylguanine residues when they are in Z-DNA (40). Studies using mammalian enzymes have not yet been performed, but the results may be similar. Clearly, inability of repair enzymes to act on methylated guanines in non-B DNA sequences could greatly contribute to the mutagenic/carcinogenic potential of these lesions. Acknowledgements This investigation was supported by grant MT-10491 from the Medical Research Council of Canada, and by the Ontario Cancer Treatment and Research Foundation.
References l.MUligan,J.R., Catz-Biro.L., Hirani-Hojatti.S., and Archer,M.C. (1989) Methylation of DNA by three W-nitroso compounds: evidence for sequence specific methylation by a common intermediate. Chem.-Biol. Interactions, 72, 175-189. 2. Briscoe.W.T. and Cotter,L.E. (1984) The effect of neighbouring bases on JV-methyl-A'-nitrosourea alkylation of DNA. Chem.-Biol. Interactions, 52, 103-110. 3 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. Interactions, 56, 321-331. 4. Van de Sande.J.H. and Jovin.T.M. (1982) Z-DNA, the left-handed helical form of poly[d(G-C)] in MgCI 2 -ethanol, is biologically active. EMBO J., 1, 115-120. 5.Saenger,W., Hunter,W.N. and Kennard.O. (1986) DNA conformation is determined by economics in the hydration of phosphate groups. Nature, 324, 385-388. 6. Herr.W. (1985) Diethyl pyrocarbonate: a chemical probe for secondary structure in negatively supercoiled DNA. Proc. Natl. Acad Sd. USA, 82, 8009-8013. 7. Fox.K.R. and Grigg,G.W. (1988) Diethyl pyrocarbonate and permanganate provide evidence for an unusual DNA conformation induced by binding of
950
the antitumour antibodies bleomycin and phleomycin. Nucleic Acids Res., 16, 2063-2075. 8 Kohwi-Shigematsu.T., Manes.T. and Kohwi.Y. (1987) Unusual conformational effect exerted by Z-DNA upon its neighbouring sequences. Proc. Natl. Acad. Sd. USA, 84, 2223-2227. 9. Kohwi-Shigematsu.T., Scribner,N. and Kohwi.Y. (1988) An ultimate chemical carcinogen, /V-acetoxy-2-acetylaminofluorence, detects non-B DNA structures that are reactive with chloroacetaldehyde in supercoiled plasmid DNA. Carcinogenesis, 9, 457—461. 10. Vogt.N., Marrot.L., Rousscau.N., Malfoy.B. and Leng.M. (1988) Chloroacetaldehyde reacts with Z-DNA. J. MM. Bioi, 201, 773-776. 11. Johnston,B.H. and Rich,A. (1985) Chemical probes of DNA conformation: detection of Z-DNA at nucleotide resolution. Celt, 42, 713-724. 12. SawadaishiJ., Miura.K., Ohtusuka.E., Ueda.T., Shinviki.N. and Ishizaki.K. (1986) Structure and sequence-specificity of ozone degradation of supercoiled plasmid DNA. Nucleic Acids Res., 14, 1159-1169. 13. Rio,P. and Leng,M. (1983) Preferential binding of the chemical carcinogen A'-hydroxy-2-aminofluorene to B-DNA as compared to Z-DNA. Nucleic Adds Res., 11, 4947-4956. 14. Marrot.L., Herbert.E., Saint-Ruf.G. and Leng.M. (1987) Comparison of the reactivity of B-DNA and Z-DNA with two isosteric chemical carcinogens 2-^^-acetoxyacetylaminofluorene and 3-jV,jV-acetoxyacetylamino-4,6-dimethyhdipyrido[l,2-a:3',2'-