Chem. Res. Toxicol. 1991, 4,641-654 (17) Renwick, A. G. (1989) Sulphoxides and sulphones. In Sulphur-Containing Drugs and Related Organic Compounds (Damani, L. A., Ed.) Part B, pp 133-154, Ellis Horwood, Chichester. (18) Halpert, J. R., Balfour, C., Miller, N. E., and Kaminsky, L. S. (1986) Dichloromethyl compounds as mechanism-based inactivators of rat liver cytochromes P-450 in oitro. Mol. Pharmacol. 30, 19-24. (19) Halpert, J. R., Jaw, J.-Y., and Balfour, C. (1989) Specific inactivation by l7b-substituted steroids of rabbit and liver cytochromes P-450 responsible for progesterone 21-hydroxylation. Mol. Pharmacol. 34, 139-147. (20) Ortiz de Montellano, P. R. (1986) Oxygen activation and transfer. In Cytochrome P-450, Structure, Mechanism and Biochemistry (Ortiz de Montellano, P. R., Ed.) pp 217-271, Plenum Publishing, New York. (21) Osawa, Y., and Pohl, L. R. (1989) Covalent bonding of the prosthetic heme to protein: A potential mechanism for the suicide inactivation or activation of hemoproteins. Chem. Res. Toxicol. 2, 131-141. (22) Correia, M. A., Decker, C., Sugiyama, K., Caldera, P., Bornheim, L., Wrighton, S. A., Rettie, A. E., and Trager, W. F. (1987) Degradation of rat hepatic cytochrome P-450 heme by 3,5-dicarbethoxy-2,6-dimethyl-4-ethyl-1,4-dihydropyridine to irreversibly bound protein adducts. Arch. Biochem. Biophys. 258,435-451. (23) Zemaitis, M. A., and Greene, F. E. (1976) Impairment of hepatic microsomal drug metabolism in the rat during daily disulfiram
647
administration. Biochem. Pharmacol. 25, 1355-1360. (24) Goldberg, M. T. (1987) Inhibition of genotoxicity by diallyl sulfide and structural analogues. In Anticarcinogenesis and Radiation Protection (Cerutti, P. A,, Nygaard, 0. F., and Simic, M. G., Eds.) pp 905-912, Plenum Publishing, New York. (25) Decker, C., Sugiyama, K., Underwood, M., and Correia, M. A. (1986) Inactivation of rat hepatic cytochrome P-450 by spironolactone. Biochem. Biophys. Res. Commun. 136, 1162-1169. (26) Decker, C. J., Rashed, M. S., Baillie, T. A., Maltby, D., and Correia, M. A. (1989) Oxidative metabolism of spironolactone: evidence for the involvement of electrophilic thiosteroid species in drug-mediated destruction of rat hepatic cytochrome P450. Biochemistry 28, 5128-5136. (27) Koop, D. R. (1990) Inhibition of ethanol-inducible cytochrome P450IIE1 by 3-amino-1,2,4-triazole. Chem. Res. Toxicol. 3, 377-383. (28) Gannett, P. M., Iversen, P., and Lawson, T. (1990) The mechanism of inhibition of cytochrome P450IIE1 by dihydrocapsaicin. Bioorg. Chem. 18, 185-198. (29) Brady, J. F., Xiao, F., Wang, M.-H., Li, Y., Ning, S. M., Gapac, J. M., and Yang, C. S. (1991) Effects of disulfiram on hepatic P450IIE1, other microsomal enzymes, and hepatotoxicity in rats. Toxicol. Appl. Pharmacol. 108, 366-373. (30) Guengerich, F. P., Kim, D.-H., and Iwasaki, M. (1991) Role of human cytochrome P-450 IIEl in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 4, 168-179.
Positional Effects on the Structure and Stability of Abbreviated H-ras DNA Sequences Containing 0 ‘-Methylguanine Residues at Codon 12 Ronald E. Bishop and Robert C. Moschel* Chemistry of Carcinogenesis Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, P.O. Box B , Frederick, Maryland 21702 Received December 26, 1990 Activation of the H-ras protooncogene in rats by methylating carcinogens results from a G-to-A transition mutation a t the second position of codon 1 2 (GGA), presumably due to formation of an @-methylguanine (m6G) a t this position. A similar transition at the fist position of codon 12 appears not to occur in vivo. T o study the possible structural basis for this bias in mutation, we synthesized a series of 11-base H-ras sequences [e.g., 5’-d(CGCTG*G*AGGCG)-3’ and two complementary strands] containing an m6G a t the first, second, or both positions of codon 12 (i.e., G* = m6G). The results of solution chemical studies indicated that the individual strands formed stable hairpin structures among which that containing m6G a t the second position of codon 1 2 was most stable. Further, the DNA duplex with m6G a t the second position was significantly more stable than that with m6G a t the first position, and under certain conditions, it was more stable than the unmodified duplex as well. I t is possible that such a difference in stability might lead to more ready recognition of an m6G a t the first position by repair proteins, and this could contribute to the apparent site specificity of mutation by methylating carcinogens a t codon 1 2 of the H-ras gene,
Introduction The production of 06-methylguanine residues by methylating carcinogens at codon 12 (GGA) of the rat H-ras gene is presumed responsible for inducing the G-to-A transition mutations that convert the normal gene to a highly transforming oncogene (1-4). Interestingly, only the second guanine of codon 12 is mutated. It is unclear if this results from a preferential reaction of the second guanine with the methylating carcinogen or if repair of an Os-methylguanineresidue at the first position of codon 12 is more efficient than repair at the second position. Knowledge of the DNA structural perturbations produced by Os-methylguanine (m6G) residues (Figure 1) should further our understanding of how these adducts are rec-
ognized by repair proteins specific for this damage (5-7) as well as how these modified guanines code for thymine incorporation during DNA replication (8-14). To model the possible effects of 06-methylguanine substitution on the structure of the H-ras gene, we have synthesized four undecamer (11-mer) oligodeoxyribonucleotides (oligonucleotides) of type 1 (Figure l ) , which are abbreviated versions of the H-ras sequence encompassing codon 12 (GGA) and spanning the third base of codon 10 through the first base of codon 14. We have compared the solution properties of the unmodified single-strand oligonucleotide 1 with those of the analogues containing an 06-methylguanine residue at the first or second position, or both the first and second positions, of the modeled codon 12 [i.e.,
0893-228~/91/2704-0647$02.50/0 0 1991 American Chemical Society
Bishop and Moschel
648 Chem. Res. Toxicol., Vol. 4, No. 6,1991 1
5‘ - d(CGCTGGAGGCG) - 3‘
1(5-m6G)
5‘ - d(CGCThGAGGCG)- 3‘
1(6-m6G)
5’ - d(CGCTGhAGGCG) - 3‘
1(5,6-di-m6G)
2
3
5’- d(CGCTbbAGGCG)
5‘
- 3’
- d(CGCCTCCAGCG) - 3’
5’ - d(CGCCll-rAGCG)
-3
OCH,
I
m6G
Figure 1. Synthetic DNA sequences and structure of 06methylguanine. 1(5-m6G), 1(6-m6G), and 1(5,6-di-m6G), respectively] as well as the properties of the duplexes of these sense strands when they are paired with oligonucleotide 2 or oligonucleotide 3 (Figure 1). T h e results of these studies indicate a significant positional effect of P - m e t h y l g u a n i n e substitution o n t h e structure a n d stability of these abbreviated H-ras sequences.
Materials and Methods
methylguanine residues were redissolved in 10 mL of methanol/ 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (9:l)and allowed to stand at room temperature for 8 days. The methanol was removed under reduced pressure, and the DNA was precipitated by the addition of 20 mL of diethyl ether with vigorous mixing. After a second ether wash, the recovered solid was dried for chromatographic purification. For this, each oligomer was dissolved in 0.1 M triethylammonium acetate (TEAA) solution, pH 7 , and chromatographed on a 10 mm X 25 cm column eluted with a linear gradient of 10-40% acetonitrile in 0.1 M TEAA over 60 min a t a flow rate of 3 mL/min (17). The retention time for all group the oligonucleotides bearing a 5’-0-(4,4’-dimethoxytrityl) was in the range of 40-45 min. Pooled samples were then lyophilized and detritylated by treatment with 80% acetic acid for 15 min followed by coevaporation with ethanol. Samples were then redissolved in TEAA solution and chromatographed on a 10 mm X 25 cm column using a gradient of 5-4070 acetonitrile in 0.1 M TEAA over 60 min a t a flow rate of 3 mL/min. Under these conditions, all nontritylated oligodeoxyribonucleotideseluted between 18 and 20 min. Samples were then lyophilized and rechromatographed using a gradient of 8-20% acetonitrile in 0.1 M TEAA over 60 min a t 3 mL/min. Retention times for the oligonucleotides were in the range of 17-19 min in all cases. Oligonucleotides were converted from the triethylammonium salt to the sodium salt by one ethanol precipitation from a 0.3 M ammonium acetate/O.Ol M magnesium acetate solution followed by three ethanol precipitations from 0.3 M sodium acetate solution. Samples were then dried by lyophilization and stored a t -20 “C. Gel Electrophoresis. Native polyacrylamide gel electrophoresis was w i e d out on 30 cm x 40 cm X 0.4 mm gels consisting of 20% acrylamide/N,”-methylenebis(acry1amide) (19:l) run in 0.045 M Tris-borate, pH 8.25, and 1.25 mM EDTA. Samples of 5’-labeled DNA (0.7-0.9 pmol/lane) were electrophoresed a t 4 “C. Gels were exposed to X-ray film, and the bands in each of the autoradiographs were quantified using an Ultroscan XL laser densitometer (Pharmacia LKB, Uppsala, Sweden). Nucleoside Composition Analysis. Oligonucleotides (0.25-0.35 ODzw unit) were treated with 0.1 unit of phosphodiesterase and 1.0 unit of alkaline phosphatase in 0.2 mL of 0.05 M Tris-HC1 and 0.005 M MgCl,, pH 8.2, for a minimum of 3 h a t 37 “C. Resulting samples were chromatographed as described before (17), and peak areas for the liberated nucleosides were converted to nanomoles of nucleoside by comparison with calibration curves generated with known amounts of authentic standards. From data on the nanomoles of liberated nucleosides, the molar absorptivities were calculated for these oligonucleotides. Thermal Denaturation Studies. Temperature-dependent absorption changes for samples of single strands or duplexes were measured in 0.01 M NaH2P04,pH 7.0,O.OOI M EDTA, and either 0.10,0.20,0.50, 1.0, or 5.0 M NaCl. For studies involving duplexes, appropriate amounts of each complementary oligomer were dissolved together and the resulting solutions were heated to 90 O C and then cooled to 40 “C over 2 h. After 1 h they were cooled slowly to room temperature before temperature-dependent absorption changes were measured. Measurements were made a t 280 or 287 nm. The heating rate for these studies was 0.25 or 0.50 “C/min. Values of T , were determined from each curve by standard methods (18-21). Thermodynamic parameters were calculated from van’t Hoff plots of T,-l vs In (CT/4), where CT is the total concentration of single-strand oligonucleotides (20, 21). Lines were fit by least-squares linear regression.
Reagents and solvents were from Aldrich Chemical Co., Inc., Milwaukee, WI, American Burdick & Jackson, Muskegon, MI, or J. T. Baker Chemical Co., Phillipsburg, NJ. Standard nucleoside phoaphoramiditesand other DNA synthesis reagents were from Applied Biosystems, Inc., Foster City, CA. Unmodified 2’-deoxyribonucleosideswere from Pharmacia LKB Biotechnology, Inc., Piscataway, NJ. Snake venom phosphodiesterase (type VII from Crotalus atrox) and bacterial alkaline phosphatase (type I11 from Escherichia coli) were from Sigma Chemical Co., St. Louis, MO. T, polynucleotide kinase was from New England Biolabs, Beverly, MA, and [?--]ATP was from Amenham Corp., Arlington Heights, IL. High-pressure liquid chromatography (HPLC) was carried out on Beckman Ultrasphere ODS columns (4.6 mm X 25 cm or 10 mm X 25 cm, 5-pm particle size) using a Waters Associates HPLC system or an LDC Constametric I1 system. Ultraviolet absorbance of the column effluent was transmitted to a Hewlett-Packard 3350 laboratory automation system for electronic integration of peak areas. Temperaturedependent absorption changes for DNA solutions were measured on a Gilford Model 250 spectrophotometer equipped with a Model 2527 thermoprogrammer and heat pump controlled cuvette compartment. Circular dichroism spectra were recorded on a Jasco 5-500 A spectropolarimeter with an IF-50011 computer interface. DNA Synthesis and Purification. Oligodeoxyribonucleotides were synthesized on a IO-pmol scale using an Applied Biosystems, Results Inc., Model 380B DNA synthesizer and recommended synthesis cycle. 06-Methyl-5’-0-(4,4’-dimethoxytrity1)-~-i~ob~tyryl-2’-All oligonucleotides illustrated in Figure 1 were syndeoxyguanosine (15, 16) was converted to the corresponding thesized using phosphite triester chemistry on a 10-pmole 3’-[ 0-(2-cyanoethyl) NJV-diisopropylphosphoramidite]using an scale with a n automated DNA synthesizer. Suitably proin situ approach described earlier (17). The resulting phosphotected 06-methyl-2’-deoxyguanosine (15,161 was converted ramidite was allowed to couple for 5 min. Coupling efficiencies to a 3‘[ 0-(Zcyanoethyl) N,N-diisopropylphosphoramidite] were determined through a 4,4’-dimethoxytritylcation assay. At derivative using our previously described in situ activation the end of synthesis, the terminal 5’-0-(4,4’-dimethoxytrityl) group approach (17) and was used immediately for DNA synwas not removed. Oligonucleotides were cleaved from the solid thesis. Coupling efficiencies for t h e 06-substituted guanine support using standard NH40H treatment and were further phosphoramidite were 87% or higher in all cases. Cleavage deprotected by treatment with NH40H a t 55 OC for 8 h. Samples were then evaporated to dryness. Oligomers containing 06of t h e D N A segments from t h e immobilized synthesis
@-Methylguanine Effects on Models of H-ras D N A
T, and Hyperchromicity Data for Oligodeoxyribonucleotides T,, "C for NaCl hyperchromicity, % concn for NaCl concn oligomer 0.1 M 1.0 M 5.0 M 0.01 M 1.0 M 5.0 M 1 68.5 67.2 50.7 30.0 26.5 25.1 l(5-m6G) 63.5 62.0 47.0 33.7 29.5 11.7 1(6-msG) 71.2 69.5 52.7 24.0 20.9 7.7 1(5,6-di-m6G) 66.2 65.3 54.0 24.7 20.8 15.9 2 57.2 59.2 45.5 18.2 16.1 7.4 3 63.7 64.8 -" 9.2 10.3 -b Table 11.
Table I. Nucleoside Composition of Synthetic
Oliaodeoxyribonucleotides nucleoside composition found" (nucleoside composition expected) oligonucleotide dCyd dGuo dThd dAdo msG 1.00 NP' 1 2.98 6.01 1.01 (3) (6) (1) (1) 1 (5-m"G) 2.98 5.02 1.00 0.98 1.02 (3) (5) (1) (1) (1) 4.98 0.99 0.98 1.08 1(6-msG) 2.97 (1) (1) (3) (5) (1) 1(5,6-di-m6G) 2.98 4.15 1.01 0.99 1.87 (1) (2) (3) (4) (1) 2 6.03 2.96 1.01 1.00 NP (1) (6) (3) (1) 3 4.00 2.99 3.00 1.01 NP (4) (3) (3) (1)
Chem. Res. Toxicol., Vol. 4, No. 6, 1991 649
t,M,
8.33 8.21 8.21 8.03 7.86 7.93
a Results are the average of four determinations. Calculated from the total number of nanomoles of nucleoside liberated in a digest of a known ODzsovalue when ODzsois measured at 25 O C on native samples. The clsD values can be increased by much as 14% if ODzsomeasurements are made on denatured samples that have been heated to 90 "C and cooled quickly on ice. The lower values reported above reflect the hypochromicity associated with the stable hairpin secondary structures for these oligonucleotides (see text). CNP,not present.
support and removal of the majority of protecting groups were accomplished using ammonium hydroxide treatments. For the carcinogen-modifiedDNA, this treatment was followed by exposure of the samples to methanol/ 1,8-diazabicyclo[5.4.0]undec-7-ene(91). This latter step is required for complete removal of the M-isobutyryl protecting group present on the 06-substituted 2'-deoxyguanosine residues in the modified DNA segments (5,16, 22). All the oligonucleotides of Figure 1were purified by reversed-phase high-pressure liquid chromatography to afford approximately 100 ODzsounits of modified DNA segment per 10-pmol synthesis, compared with 150 ODzm units of unmodified DNA segment per synthesis. Samples were characterized by digestion to their component 2'deoxyribonucleosideswith snake venom phosphodiesterase and bacterial alkaline phosphatase, and the amounts of liberated nucleosides were analyzed using a liquid chromatographic system that we developed earlier (17). The results of these analyses are presented in Table I together with the molar absorptivities for these oligomers. These data show agreement between the expected and experimentally determined nucleoside compositions and confirm that the oligonucleotides are free of significant contamination by incompletely deprotected nucleosides or possible nucleoside contaminants produced during synthesis and deprotection steps (17, 22, 23). Our studies of the solution properties of the various abbreviated H-ras sequences (Figure 1) began with an examination of the melting profiles for the unmodified strands 1, 2, and 3 and the modified strands 1(5-m6G), 1(6-m6G),and 1(5,6-di-m6G)in 0.01 M NaH2P04and 0.001 M EDTA, pH 7.0, as a function of NaCl concentration. Representative profiles for these oligonucleotides are shown in Figure 2, panels A and B. These data illustrate that dilute solutions (i.e., 2 X lo6 M) of all oligonucleotides other than 3 exhibited relatively high T, values and considerable hyperchromicity on denaturation. Such properties are indicative of their ability to assume stable secondary structures under these solvent conditions. A taand accompanying hyperchromicity bular summary of T,,, data for these single strands in 0.1, 1.0, and 5.0 M NaCl solution is presented in Table 11. As indicated in the table, the observed hyperchromicity and T, data in 1.0 M NaCl are very similar to those in 0.1 M NaC1, while data for these
" Reproducible melting was not observed for oligomer 3 in 5.0 M NaC1. b A 7.8% decrease in absorbance at 280 nm was observed as the temperature was increased from 5 to 100 OC. Chart I. Hairpin Structures for Representative DNA Segments G G A T G C-G G-C C-G
G'A T G C-G G-C C-G
d G A T G C-G G-C C-G
1
1(6-m6G)
1(5,6-dCm6G)
oligonucleotides in 5.0 M NaCl indicate that this high salt concentration has a destabilizing effect on the ordered structures in all cases. While stable secondary structures could result from either interstrand associations (e.g., cruciform or multiplex structures) or intrastrand associations (hairpin loops) under these conditions, the latter structures are known to be favored in dilute solution at low ionic strength (24-28). Furthermore, the melting behavior of oligonucleotidesthat adopt hairpin structures is known to be fairly insensitive to changes in ionic strength over a low range similar to that which we have studied (i.e., 0.1-1.0 M NaC1) (29). Thus, the data of Figure 2A,B together with that of Table I1 suggest that all single strands 1 and 2 form hairpin loops in the 0.1-1.0 M NaCl range. Examples of these structures are shown in Chart I. Interestingly, the various T, data indicate that oligonucleotide 1(6-m6G), having an 06methylguanine residue at the midpoint of the sequence (i.e., at the second position of codon 12 in these abbreviated models of the H-ras sequence), formed the most stable hairpin structure, followed by the unsubstituted strand 1 and the strand containing two adjacent P-methylguanine residues [Le., 1(5,6-di-msG)] (Chart I). The hairpin adopted by 1(5-m6G)appeared to be less stable than that of the "isomer" 1(6-m6G)or the doubly substituted 1(5,6di-msG). Complementary strands 2 and 3 also appeared to form hairpin structures. The lower hyperchromicity associated with their thermal transitions is no doubt related to their having pyrimidine-rich rather than purinerich loops as in type 1 oligonucleotides. Thus, for these single strands, the presence of an m6G residue at the sixth position has a marked stabilizing effect on all the hairpin structures. The stability of the intramolecular hairpin structures adopted by type 1 oligonucleotides might be expected to disfavor formation of intermolecular duplexes between strands 1 and 2. However, when samples of the type 1 oligonucleotides were mixed with an equimolar amount of 2 at high temperature and the mixture was allowed to cool slowly to 25 "C or less, intermolecular duplexes formed readily. Representative normalized melting profiles for these are illustrated in Figure 2, panels C and D. Figure 2C illustrates that, in 0.1 M NaCl, the unsubstituted duplex [l + 21 exhibited simple two-state duplex to single strand thermal transitions. The duplexes containing
Bishop and Moschel
650 Chem. Res. Toxicol., Vol. 4 , No. 6, 1991
A
B
0.10M NaCl 1 .M
0.w
0.10 M NaCl
1.30
1.20
-
1.10
-
( 1 / 1 1 1 1 1 1 1 ( o
io
2030
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50
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70
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-
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2 Bo
80
1w
0
1
0
2
0
3
0
4
0
5
O
w
7
0
~
w
I)
Temperature ("C)
Figure 2. Absorbance versus temperature profiles for synthetic DNA segments. Profiles were determined at single-strand concentrations of 20 pM in 0.01 M sodium phosphate and 0.001 M EDTA, pH 7.0, at the indicated NaCl concentration. Absorbance was monitored at 280 nm. P-methylguanine residues exhibited broader major transitions and also showed a minor transition near 30 "C. Such profiles could indicate a low-temperature transition of double helix to single-strand hairpins followed by melting of the hairpin structures to random coils (29). However, if only the minor transitions were reflecting duplex dissociation, then only their T, values would be expected to increase with increasing oligonucleotide concentration. T, values for the major transition would be expected to be independent of oligonucleotide concentration (29). Table I11 contains T, data for these duplexes as a function of NaCl and oligonucleotide concentration, and these data indicate that, for all duplexes involving strands of types 1 and 2 in both 0.1 and 0.2 M NaCl as well as for [l 21 in 0.5 M NaC1, the T, values for both the major and minor transitions (transition 2 and transition 1, respectively, Table 111) increase with increasing oligonucleotide concentration. Figure 3 shows van't Hoff plots for the major transition (transition 2, Table 111) in 0.2 M NaCl. Related plots for the minor transitions are also linear although somewhat more scattered. The negative slopes for the linear plots confirm the concentration dependence for these transitions, which is indicative of duplex dissociation under these conditions. Thermodynamic parameters calculated from the slopes of these van't Hoff plots are presented in Table IV. The enthalpy values calculated for these transitions are in the range expected for such G-C-rich duplexes (30). Further, our data for [ 1 + 21 and [ 1(5-msG) + 21 in 0.2 M NaCl indicate that the
+
I :
2.901
- 12.5
1
I
- 12.0
-11.5
I
-11.0
I"(?)
Figure 3. Concentration dependence of T,,, for oligonucleotide duplexes in 0.2 M NaCl. Lines were fit by least-squares linear regression analysis. presence of an 06-methylguanine residue in place of the first guanine in these modeled H-ras sequences decreases the enthalpy for dissociation by 7 kcal/mol, which is near the 8-11 kcal/mol range expected in 0.15 M NaCl on the basis of the data of Gaffney and Jones (21). However, enthalpy values for the dissociation of the [ 1(6-msG)+ 21 duplex were higher than for either [ 1(5-meG)+ 21 or [ 1 + 21 under these salt conditions, indicating that an 08methylguanine in place of a guanine at the second position of these modeled H-ras duplexes can actually stabilize the duplex relative to the unmodified parent. This resembles the stabilizing effect observed for an m6G at the midpoint
@-Methylguanine Effects on Models of H-ras DNA Table 111.
duplex [ I + 21
CT,”
Tm, “C
M
pM
0.10
19.9 30.7 40.4 61.0 10.2 20.4 30.4 40.7 50.3 60.5 20.1 39.4 59.6 18.4 19.9 20.6 17.6 19.3 21.3 41.6 52.9 60.9 20.1 30.0 40.4 52.6 59.8 19.4 40.3 61.7 20.1 23.0
transition 1 transition 2b -C 61.5 61.7 62.0 62.2 61.8 63.7 64.2 64.9 65.5 65.8 66.1 67.2 68.2 66.3 57.2 69.7 66.2 53.0 63.0 27.0 64.8 30.0 65.3 31.0 66.0 33.5 62.3 32.0 63.0 34.5 37.0 64.0 64.5 36.0 65.0 39.0 61.7 61.8 60.9 57.2 43.0
0.50
+ 31 (1(5-m6C) + 21
T, Data for Oligodeoxyribonucleotide Duplexes
NaCl concn,
0.20
1.00 5.00 0.10 1.00 5.00 0.10
0.20
0.50 1.00 5.00
Chem. Res. Toxicol., Vol. 4, No. 6, 1991 651
duplex [ 1(6-m6G) + 21
NaCl concn,
CT,”
M
pM
0.10
20.5 41.9 51.5 58.4 19.8 30.2 39.9 51.1 59.9 20.3 39.7 59.7 19.5 21.4
0.20
0.50 1.00 5.00 0.10
[1(5,6-di-m6G)+ 21
0.20
0.50 1.00 5.00 0.10 1.00 5.00
[1(5,6-di-m6G)+ 31
OTotal single strand Concentration. *The major transition in all cases is transition 2.
c(-)
21.1
42.5 54.0 59.5 20.3 29.4 40.5 51.9 60.4 20.8 39.4 59.7 19.0 20.5 22.0 18.6 20.9
Tm, “C transition 1 transition 2b 30.2 67.0 32.5 68.5 33.5 69.5 34.5 69.8 34.0 67.1 38.7 67.5 36.0 68.0 37.5 69.0 40.0 69.3 35.0 65.5 36.3 65.8 65.6 37.0 61.3 48.0 22.5 65.0 23.0 65.5 32.4 65.6 65.7 63.1 65.0 65.8 39.2 66.7 41.0 67.3 35.5 64.3 64.0 64.0 20.0 60.7 44.0 65.7 65.0 49.5
Not present.
Table IV. Thermodynamic Values for Duplex to Single Strand Transitions for Oligodeoxyribonucleotide Duplexes AHo, kcal/ mol AS”, kcal/(K.mol) AGO (25 “C), kcal/mol duulex NaCl concn. M transition 1 transition 2 transition 1 transition 2 transition 1 transition 2 11 + 21 0.20 -a 100.2 0.27 18.7 0.50 121.4 0.33 23.1 [1(5-m6C) + 21 0.10 33.1 83.6 0.09 0.22 7.4 16.7 0.20 33.7 93.2 0.09 0.25 8.0 17.6 [1(6-m6C)+ 21 0.10 48.0 87.7 0.13 0.23 8.0 18.0 0.20 48.7 109.3 0.13 0.30 8.8 20.7 [1(5,6-di-m6G)+ 21 0.20 62.5 0.16 14.3 (-) Not present.
of the single-strand hairpins (see above). In light of these observations, it remains unclear what is responsible for the minor transitions observed with the modified duplexes. These may reflect a premelting due to dissociation of protonated G C base pairs (31-33) or perhaps a thermally induced “unbending” (34) prior to dissociation of the complementary strands. Additionally, the existing data do not enable us to determine if dissociation of these duplexes proceeds directly to random coils or if single-strand hairpins are formed first as intermediates. Additional experiments would be required to test these possibilities and to test for the possible origins of the minor transitions suggested above. In 0.5 M NaCl concentrations, only the T, values for duplex [ l + 21 increase with increasing strand concentration (Tables I11 and IV). The reasons why the modified duplexes fail to exhibit similar behavior are unclear at present. Furthermore, only duplex [1 + 21 exhibits a higher T , with increasing NaCl concentration over the range 0.1-1.0 M. T , values for the major transitions exhibited by the modified duplexes are little influenced or actually decrease somewhat over this range. The modified duplexes are apparently maximally stabilized in 0.1-0.2
M NaCl solutions. In 5.0 M NaCl solution, all duplexes exhibit a markedly lower T, relative to values at lower NaCl concentrations, indicating destabilization of the duplexes at high salt as we observed with the various single strands. Under all conditions studied, however, the modified duplex [1(6-meG)+ 21 exhibited a higher T, than that of [ 1(5-msG)+ 21, which was usually very close to or greater than that exhibited by duplex [1(5,6-di-m6G) 21. However, the greater sensitivity of T, values for [ 1 21 to changes in NaCl concentration leads to significant changes in the overall stability order for these four duplexes with changing salt concentration. For example, in 0.1 M NaCl and at duplex concentrations near 10 pM,the T , value for [ l 21 is lower than that for the modified duplexes. Its T , value falls between that for [ 1(6-m6G) + 21 and [1(5-m6G) 21 in 0.2 M NaC1, while in 0.5 and 1.0 M NaCl its T, is higher than those for the other three modified duplexes. Thus, the ranking of stability of these H-rus models is a complex function of oligonucleotide and salt concentration. Our studies of the melting behavior of duplexes [l 31 or [1(5,6-di-msG)+ 31 (Table 111) are more limited. This stems from data obtained from the electrophoretic mobility and circular dichroism spectra for
+ +
+
+
+
Bishop and Moschel
652 Chem. Res. Toxicol., Vol. 4, No. 6, 1991
I
3.0
-
1.0
-
B
2.0 1.o 0
-1 .o -2.0
-2.0 -3.0
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Wavelength (nm)
Figure 4. Circular dichroism spectra of synthetic DNA duplexes as a function of NaCl concentration. Spectra were determined for solutions containing 0.01 M sodium phosphate, O.OOO1 M EDTA, pH 7.0, and (-) 0.1 M NaCl, (-.-) 1.0 M NaCl, or ( - - - ) 5.0 M NaCl. these duplexes (see below), which indicate that type 1 oligonucleotides do not associate very strongly with complementary strand 3. The T, data presented for [ 1 31 and [1(5,6-di-msG) + 31 (Table 111) probably reflect a composite of the melting of individual hairpins in the presence of the respective duplex. Circular dichroism (CD) spectra were recorded for all duplexes in buffered solution containing 0.01, 0.1, 1.0, and 5.0 M NaCl (Figure 4). While all exhibited the spectrum of B-form DNA in low-salt solution, duplexes [l + 21, [1(5-msG) + 21, [1(6-msG) + 21, and to a lesser exten [ l (5,6-di-m6G)+ 21 exhibited a transition to the C-form with increasing NaCl concentrations as evidenced by the decrease in the positive band in the 270-280-nm region of their spectra (i.e., Figure 4, panels A, C, D, and E, respectively). This is typical of double-stranded DNA samples (35). Interestingly, the increase in negative ellipticity in the 240-260-nm region exhibited by the unsubstituted duplex [ 1 + 21 with increasing salt concentration was also seen in the case of the [1(6-msG) + 21 duplex, but not in the spectra for [1(5-m6G)+ 21. This observation points to structural features shared by the unmodified duplex and that containing msG in the second position of H-ras codon 12 which are not found in the other modified duplexes.
+
The spectra for duplexes containing two G.T mismatches ([ 1 + 31) (Figure 4B) or two m6G.T pairs ([ 1(5,6-di-m6G) + 31) (Figure 4F) showed little change with increasing NaCl concentration. Very similar behavior was noted in the CD spectra of the individual strands 1, 1(5,6-di-msG), and 3 in response to increasing salt concentration (not shown), suggesting that duplexes composed of strands 1 and 3 are substantially weaker than those involving 1 and 2 and may form to only a limited extent in dilute solution. These conclusions are supported by studies of the electrophoretic mobility of the various 32P-radiolabeledduplexes on polyacrylamide gels under nondenaturing conditions. A representative electrophoretogram is shown in Figure 5. In these experiments, little or no evidence for duplex formation was observable for 1:l mixtures of 1 and 3 (lane 5, Figure 5) or 1(5,6-di-m6G) and 3 (lane 8). In contrast, all 1:l mixtures of the oligonucleotides 1 and 2 exhibited significant amounts of duplex formation, as indicated by the slower migrating bands (36)present in lanes 2-4,6, and 7 (Figure 5). The mixture of the unsubstituted strands 1 and 2 showed the strongest interstrand association under these conditions, with more than 90% of the radioactivity being present in the more slowly migrating bands (lanes 2 and 3). Approximately 70% of the radio-
@-Methylguanine Effects on Models of H-ras DNA 1
2
3
4
5
6
7
8
9
C
..-Eh0 0
L
I
Figure 5. Native polyacrylamide gel electrophoresis of radiolabeled DNA duplexes. Lanes 1and 9 contained the individual strands 1and 2,respectively. Lane 2 , l + 2 (equimolar amounts); lane 3 , l + 2 (1:1.3); lane 4,[1(6-m6G) + 21;lane 5, [ l + 31;lane 6, [1(5,6di-m%) + 21;lane 7,[1(5-m%) + 21;lane 8,[1(5,6di-m’%)
+ 31.
activity for duplex [ 1(6-m6G)+ 21 migrated in this region (lane 4). Approximately 65% duplex was exhibited by [ 1(5m6G)+ 21 (lane 7) while the mixture of 1(5,6-di-m6G) and 2 was approximately 20% duplex (lane 6). Although conditions for these electrophoresis experiments differed significantly from those used in the melting studies, the electrophoretograms confirm that oligonucleotidesof types 1 and 2 form duplex structures even under very dilute conditions. While these can dissociate to single strands under the electrophoretic conditions, the data do not establish whether these are random coils or hairpins. Nevertheless, taking together these observations along with the CD spectral changes accompanying increasing NaCl concentration (Figure 4) and the data of Tables I11 and IV, the order of duplex stability in 0.1-0.2 M NaCl solutions would be as follows: [1(6-m6G)+ 21 2 [1 + 21 > [1(5-m6G)+ 21 > [1(5,6-di-m6G)+ 21 > [1(5,6-di-m6G)+ 31 > [1 + 31.
Discussion Our data demonstrate that these abbreviated H-ras sequencesundergo significant structural changes when an @-methylguanine residue is incorporated in place of a normal guanine in the codon 12 (GGA) triplet. In particular, replacement of the second guanine with an m6G residue leads to a more stable single-strand hairpin structure than is observed for the unmodified strand 1 or the modified analogues having the m6G at the fmt position or first and second positions of the GGA sequence. In double-stranded structures, the incorporation of an 06methylguanine residue at the first base of the GGA triplet disrupts the stability of the parental duplex, as expected. Remarkably, the most stable modified duplex in solution near physiological ionic strength (37)results from incorporation of the V-methylguanine at the second position of codon 12 in these models. Structural details accounting for the unusual stability of this modified duplex are not available as yet. However, in view of the CD spectral characteristics shared by [l + 21 and [1(6-m6G) + 21, it seems likely that these structures are more similar to one another than are those for [ l + 21 and [1(5-m6G)+ 21. Using related although longer sequences, Voigt and Topal (38) recently demonstrated that substitution of an 06methylguanine residue in place of the first guanine of the GGA triplet led to greater backbone distortion and a lower
Chem. Res. Toxicot., Vol. 4, No. 6, 1991 653
T, than was observed for @-methylguanine substitution at the second position. The suggestion (38)that these T m differences were not observed previously (I7)because of the salt concentrations used in the T, determinations is probably correct since we have shown here that T, differences as a function of V-methylguanine position in these modeled H-ras sequences are highly dependent on NaCl concentration. In light of all these observations, it seems reasonable that the greater instability or more pronounced structural distortions imparted to H-ras sequences by 06-methylguanine substitution at the first position rather than the second position of codon 12 could lead to more ready recognition and repair of the lesion at the first position (5,38,39) and this could contribute to the apparent site specificity of mutation at d o n 12 of the rat H-ras gene. Acknowledgment. This work was supported by the National Cancer Institute, DHHS, under Contract N01CO-74101 with ABL. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Govemment. References (1) Sukumar, S.,Notario, V., Martin-Zanca, D., and Barbacid, M. (1983)Induction of mammary carcinomas in rats by nitroso-methylurea involves malignant activation of H-ras-1 locus by single point mutations. Nature (London) 306,658-661. (2) Zarbl, H.,Sukumar, S., Arthur, A. V., Martin-Zanca, D., and Baracid, M. (1985)Direct mutagenesis of H-ras-1 oncogenes by N-nitroso-N-methylurea during initiation of mammary carcinogenesis in rats. Nature (London) 315,382-385. (3)Wang, Y., You, M., Reynolds, S. H., Stoner, G. D., and Anderson, M. W. (1990)Mutational activation of the cellular Harvey ro5 oncogene in rat esophageal papillomas induced by methylbenzylnitrosamine. Cancer Res. 50, 1591-1595. (4) Mitra, G., Pauly, G. T., Kumar, R., Pei, G. K., Hughes, S.H., Moschel, R. C., and Barbacid, M. (1989)Molecular analysis of @-substituted guanine-induced mutagenesis of ras oncogenes. Proc. Natl. Acad. Sci. U.S.A. 86,8650-8654. (5) Topal, M. D., Eadie, J. S., and Conrad, M. (1986)@-Methylguanine mutation and repair is nonuniform. Selection for DNA most interactive with Os-methylguanine. J. Biol. Chem. 261, 9879-9885. (6) Dolan, M. E.,Oplinger, M., and Pegg, A. E. (1988) Sequence specificity of guanine alkylation and repair. Carcinogenesis 9, 2139-2143. (7) Pegg, A. E. (1990)Mammalian 08-alkylguanine-DNA alkyltransferase: Regulation and importance in response to alkylating carcinogenic and therapeutic agents. Cancer Res. 50,6119-6129. (8)Loveless, A. (1969)Possible relevance of @-alkylation of deoxyguanosine to the mutagenicity and carcinogenicity of nitrosamines and nitrosamides. Nature (London) 223,206-207. (9)Mehta, J. R.,and Ludlum, D. B. (1989)Synthesis and properties of 06-methyldeoxyguanylicacid and ita copolymers with deoxycytidylic acid. Biochin. Biophys. Acta 521,770-778. (10) Abbott, P. J., and Saffhill, R. (1979)DNA synthesis with methylated poly(dC-dG) templates. Evidence for a competitive nature to miscoding by Os-methylguanine. Biochim. Biophys. Acta 562,51-61. (11)Singer, B., Chavez, F., Goodman, M. F., Easigman, J. M., and Dosanjh, M. K. (1989)Effect of 3’flanking neighbors on kinetics of pairing of dCTP or dTTP opposite Os-methylguaninein a defined primed oligonucleotide when Escherichia coli DNA polymerase I is used. Proc. Natl. Acad. Sei. U.S.A. 86, 8271-8274. (12)Toorchen, D., and Topal, M. D. (1983)Mechanisms of chemical mutagenesis and carcinogenesis: effects on DNA replication of methylation a t the @-guanine position of dGTP. Carcinogenesis 4, 1591-1597. (13)Loechler, E.L.,Green, C. L., and E s s i i , J. M. (1984)In vivo mutagenesis by Os-methylguanine built into a unique site in a viral genome. h o c . Natl. Acad. Sei. U.S.A. 81,6271-6275. (14)Basu, A. K., and Essigman, J. M. (1988)Site-specifically modified oligodeoxynucleotides as probes for the structural and bio-
654 Chem. Res. Toxicol., Vol. 4 , No. 6,1991 logical effecta of DNA-damaging agents. Chem. Res. Toxicol. 1, 1-18. (15) Gaffney, B. L., and Jones, R. A. (1982) Synthesis of @-alkylated deoxyguanosine nucleosides. Tetrahedron Lett. 23, 2253-2256. (16) Kuzmich, S., Marky, L. A,, and Jones, R. A. (1983) Specifically alkylated DNA fragments. Synthesis and physical characterization of d[CGC(@Me)GCG] and d[CGT(@Me)GCG]. Nucleic Acids Res. 11,3393-3404. (17) Pauly, G. T., Powers, M., Pei, G. K., and Moschel, R. C. (1988) Synthesis and properties of H-ras sequences containing @-substituted 2'-deoxyguanosine residues at the first, second or both positions of codon 12. Chem. Res. Toxicol. 1, 391-397. (18) Albergo, D. D., Marky, L. A., Breslauer, K. J., and Turner, D. H. (1981) Thermodynamics of (dG-dC)3double-helix formation in water and deuterium oxide. Biochemistry 20, 1409-1413. (19) Marky, L. A,, and Breslauer, K. J. (1987) Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 26, 1601-1620. (20) Borer, P. N., Dengler, B., Tinoco, I., Jr., and Uhlenbeck, 0. C. (1974) Stability of ribonucleic acid double-stranded helices. J. Mol. Biol. 86, 843-853. (21) Gaffney, B. L., and Jones, R. A. (1989) Thermodynamic comparison of the base pairs formed by the carcinogenic lesion 06methylguanine with reference both to Watson-Crick pairs and to mismatched pairs. Biochemistry 28, 5881-5889. (22) Borowy-Borowski,H., and Chambers, R. W. (1979) A study of side reactions occurring during synthesis of oligodeoxynucleotides containing @-alkyldeoxyguanosine residues at preselected sites. Biochemistry 26, 2465-2471. (23) Jones, R. A. (1984) Preparation of protected deoxyribonucleosides. In Oligonucleotide Synthesis: A Practical Approach (Gait, M. J., Ed.) pp 23-34, IRL Press Limited, Oxford, U.K. (24) Scheffler, I. E., Elson, E. L., and Baldwin, R. L. (1968) Helix formation by dAT oligomers. I. Hairpin and straight-chain helices. J . Mol. B i d . 36, 291-304. (25) Wemmer, D. E., Chou, S. H., Hare, D. R., and Reid, B. R. (1985) Duplex-hairpin transitions in DNA NMR studies on CGCGTATACGCG. Nucleic Acids Res. 13, 3755-3772. (26) Summers, M. F., Byrd, R. A., Gallo, K. A,, Samson, C. J., Zon, G., and Egan, W. (1985) Nuclear magnetic resonance and circular dichroism studies of a duplex-single-stranded hairpin loop equilibrium for the oligodeoxyribonucleotide sequence d(CGCGATTCGCG). Nucleic Acids Res. 13,6375-6386. (27) Senior, M. M., Jones, R. A., and Breslauer, K. J. (1988) Influence of loop residues on the relative stabilities of DNA hairpin
Bishop and Moschel structures. Proc. Natl. Acad. Sci. U.S.A. 86, 6242-6246. (28) Germann, M. W., Kalisch, B. W., Lundberg, P., Vogel, H. AJ., and van de Sande, J. H. (1990) Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies. Nucleic Acids Res. 18, 1489-1498. (29) Marky, L. A., Blumenfeld, K. S., Kozlowski, S., and Breslauer, K. J. (1983) Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCGAATTCGCG): evidence for hairpin formation. Biopolymers 22, 1247-1257. (30) Breslauer, K. J., Frank, R., Blocker, H., and Marky, L. A. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750. (31) Williams, L. D., and Shaw, B. R. (1987) Protonated base pairs explain the ambiguous pairing properties of @-methylguanine. Proc. Natl. Acad. Sci. U.S.A. 84, 1779-1783. (32) Lamm, G., and Pack, G. R. (1990) Acidic domains around nucleic acids. Proc. Natl. Acad. Sei. U.S.A. 87, 9033-9036. (33) Sarocchi, M.-T., and Guschlbauer, W. (1973) Protonated polynucleotide structures. Sequence-dependent and protonation-sensitive metastable states in DNA premelting. Eur. J . Biochem. 34, 232-240. (34) Park, Y.-H., and Breslauer, K. J. (1991) A spectroscopic and calorimetric study of the melting behaviors of a "bent" and "normal" DNA duplex: [d(GA4T4C)IZ versus [d(GT4A4C)],. Proc. Natl. Acad. Sci. U.S.A. 88, 1551-1555. (35) Ivanov, V. I., Minchenkova, L. E., Schyolkima, A. K., and Poletayev, A. I. (1973) Different conformations of double-stranded nucleic acid in solution as revealed by circular dichroism. Biopolymers 12, 89-110. (36) Maniatis, T., Jeffrey, A., and van deSande, H. (1975) Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14, 3787-3793. (37) Ganong, W. F. (1985) Review of Medical Physiology, 12th ed., pp 13-21, Lange Medical Publications, Los Altos, CA. (38) Voigt, J. M., and Topal, M. D. (1990) @-Methylguanine and A.C and G.T mismatches cause asymmetric structural defects in DNA that are affected by DNA sequence. Biochemistry 29, 5012-5018. (39) Werntges, H., Steger, G., Riesner, D., and Fritz, H.-J. (1986) Mismatches in DNA double strands: thermodynamic parameters and their correlation to repair efficiencies. Nucleic Acids Res. 14, 3773-3790.
