Synthesis, miscoding specificity, and thermodynamic stability of oligodeoxynucleotide containing 8-methyl-2'-deoxyguanosine

Article abstract of DOI:10.1021/tx9601059

8-Methyl-2'-deoxyguanosine (8-MedG) was synthesized by reacting dG under the methyl radical generating system and incorporated into oligodeoxynucleotides using phosphoramidite techniques. The site-specifically modified oligodeoxynucleotide containing a single 8-MedG was then used as a template for primer extension reactions catalyzed by the 3'-5' exonuclease- free (exo^-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA polymerase α. Primer extension catalyzed by the exo^- Klenow fragment readily passed the 8-MedG lesion in the template while that catalyzed by pol α was retarded opposite the lesion. The fully extended products formed during DNA synthesis were analyzed to quantify the miscoding specificities of 8-MedG. Both DNA polymerases incorporated primarily dCMP, the correct base opposite the lesion, along with small amounts of incorporation of dGMP and dAMP. In addition, two-base deletion was observed only when the exo^- Klenow fragment was used. The thermodynamic stability of 8-MedG in the duplex was also studied. The duplex containing 8-MedG:dG was more thermally and thermodynamically stable than that of dG:dG. The duplex containing 8-MedG:dA was more thermodynamically stable than that of dG:dA. We conclude that 8-MedG is a miscoding lesion and capable of generating G → C and G → T transversions and deletion in cells.

Full text of DOI:10.1021/tx9601059

1278
Chem. Res. Toxicol. 1996, 9, 1278-1284
Syn th esis, Miscod in g Sp ecificity, a n d Th er m od yn a m ic
Sta bility of Oligod eoxyn u cleotid e Con ta in in g
8-Meth yl-2′-d eoxygu a n osin e
Kohfuku Kohda,*,† Hirotaka Tsunomoto,† Yasushi Minoura,‡
Kazushi Tanabe,‡ and Shinya Shibutani§
Faculty of Pharmaceutical Sciences, Nagoya City University, Tanabedori,
Mizuho-ku, Nagoya 467, J apan, Laboratory of Biochemistry, Aichi Cancer Center Research
Institute, Kanokoden, Chikusa-ku, Nagoya 467, J apan, and Department of Pharmacological
Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651
Received J une 26, 1996X
8-Methyl-2′-deoxyguanosine (8-MedG) was synthesized by reacting dG under the methyl
radical generating system and incorporated into oligodeoxynucleotides using phosphoramidite
techniques. The site-specifically modified oligodeoxynucleotide containing a single 8-MedG
was then used as a template for primer extension reactions catalyzed by the 3′f5′ exonuclease-
free (exo-) Klenow fragment of Escherichia coli DNA polymerase I and mammalian DNA
polymerase R. Primer extension catalyzed by the exo- Klenow fragment readily passed the
8-MedG lesion in the template while that catalyzed by pol R was retarded opposite the lesion.
The fully extended products formed during DNA synthesis were analyzed to quantify the
miscoding specificities of 8-MedG. Both DNA polymerases incorporated primarily dCMP, the
correct base opposite the lesion, along with small amounts of incorporation of dGMP and dAMP.
In addition, two-base deletion was observed only when the exo- Klenow fragment was used.
The thermodynamic stability of 8-MedG in the duplex was also studied. The duplex containing
8-MedG:dG was more thermally and thermodynamically stable than that of dG:dG. The duplex
containing 8-MedG:dA was more thermodynamically stable than that of dG:dA. We conclude
that 8-MedG is a miscoding lesion and capable of generating G f C and G f T transversions
and deletion in cells.
O6-Methylguanine has been reported to be a mutagenic
and carcinogenic lesion (14). However, the mutagenic
In tr od u ction
DNA damage is an initiating event in cancer (1). 1,2-
Dimethylhydrazine (DMH)1 has been known to induce
colon cancer in rodents (2) and form several methylated
DNA adducts, including N7-methylguanine and O6-
methylguanine, in tissue DNA of rodents (3-5). Such
DNA adducts are also formed by the typical methylating
carcinogens, N-methyl-N-nitrosourea (MNU) and N-
methyl-N′-nitro-N-nitrosoguanidine (MNNG) (6). MNU
and MNNG are metabolically activated to produce elec-
trophilic methanediazonium ions as a reactive species (6,
7) while DMH forms methyl radicals in vitro (8-10) and
in vivo (7) in addition to electrophilic methanediazonium
ions. Chemical (11) and biochemical (12) studies on
radical methylation reveal that the C8-position of the
guanine moiety is the most reactive site to form 8-me-
thylguanine (8-MeGua). 8-MeGua has been detected in
the liver and colon DNA of rats treated with DMH (13).
properties of 8-MedG have not been investigated. Re-
cently, various antioxidants, such as vitamin E (15) and
selenium (16), decreased the incidence of DMH-induced
colon cancer. Thus, methyl radicals induced by DMH
may play a role in mutagenesis and carcinogenesis.
We have described the synthesis of 8-MedG using the
radical methylation system and the preparation of
8-MedG-modified oligodeoxynucleotides by phosphora-
midite techniques. The site-specifically modified oligode-
oxynucleotide was used as a template in primer extension
reactions catalyzed by DNA polymerases. Miscoding
specificities of 8-MedG were determined quantitatively
using an established in vitro experimental system (17-
19). We also investigated the effect of 8-MedG lesions
on the thermodynamic stabilities to characterize the
impact of the lesions on the physicochemical properties
of DNA duplex. These results indicate that 8-MedG is a
mutagenic lesion, generating G f C and G f T trans-
versions and deletion.
* To whom correspondence should be addressed. Telephone: J apan
(81)-52-836-3408; Telefax: J apan (81)-52-834-9309; E-mail: kohda@
phar.nagoya-cu.ac.jp.
† Nagoya City University.
Exp er im en ta l P r oced u r es
‡ Aichi Cancer Center Research Institute.
Syn th esis of 8-Med G Der iva tives. Gen er a l. 1H and 13C
NMR spectra were recorded on a J EOL EX 270 or GSX 400
spectrometer (Tokyo, J apan), and chemical shifts were reported
in parts per million (ppm) using tetramethylsilane as the
internal standard. Mass spectra were measured by a J EOL DX-
300 spectrometer (Tokyo, J apan). UV spectra were recorded
on a Shimadzu UV-2100 spectrophotometer (Kyoto, J apan).
Melting points were measured with a Yanagimoto micro-melting
point apparatus (Kyoto, J apan). Merck silica gel 60 (70-230
mesh, 63-200 µm) was used for column chromatography. HPLC
§ State University of New York at Stony Brook.
X Abstract published in Advance ACS Abstracts, October 15, 1996.
1
Abbreviations: DMH, 1,2-dimethylhydrazine; 8-MedG, 8-methyl-
2′-deoxyguanosine; MNU, N-methyl-N-nitrosourea; MNNG, N-methyl-
N′-nitro-N-nitrosoguanidine; 8-oxodG, 7,8-dihydro-8-oxo-2′-deoxygua-
nosine or 8-hydroxydeoxyguanosine; 8-MeGua, 8-methylguanine; dG,
2′-deoxyguanosine; Gua, guanine; dNTP, 2′-deoxynucleoside triphos-
phate; exo-, 3′f5′ exonuclease-free Klenow fragment; pol R, DNA
polymerase R; Tm, melting temperature; ∆H°, van’t Hoff transition
enthalpy; ∆S°, entropy; ∆G°, free energy; Ct, oligonucleotide strand
concentration; PAGE, polyacrylamide gel electrophoresis; HPLC, high
performance liquid chromatography; tR, retention time.
S0893-228x(96)00105-1 CCC: $12.00 © 1996 American Chemical Society

8-Methyl-dG DNA Adduct
Sch em e 1
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1279
(482 mg). 1H NMR (Me2SO-d6) δ 10-11 (hump, 2H, 1-NH and
2-NH), 6.26 (t, 1H, J ) 7.3 Hz, 1′-H), 5.28 (d, 1H, J ) 4.3 Hz,
3′-OH), 4.84 (t, 1H, J ) 5.4 Hz, 5′-OH), 4.39 (m, 1H, 3′-H), 3.78
(m, 1H, 4′-H), 3.60-3.52 (m, 2H, 5′-CH2), 2.86-2.75 (m, 2H, 2′-
Ha and CH(CH3)2), 2.51 (s, 3H, 8-CH3), 2.15-2.06 (m, 1H, 2′-
Hb), 1.13 (d, 6H, J ) 6.6 Hz, C(CH3)2).
5′-O-(4,4′-Dim et h oxyt r it yl)-N2-isob u t yr yl-8-m et h yl-2′-
d eoxygu a n osin e (3). After N2-isobutyryl-8-methyl-2′-deox-
yguanosine (2, 476 mg, 1.35 mmol) was coevaporated with dry
pyridine (10 mL) twice, it was suspended in 10 mL of dry
pyridine. 4,4′-Dimethoxytrityl chloride (95%, 578 mg, 1.62
mmol), triethylamine (263 µL, 1.89 mmol), and 4-(dimethylami-
no)pyridine (8.3 mg, 0.068 mmol) were added to the solution,
and the mixture was stirred at room temperature for 2 h. After
10 mL of H2O was added, the product was extracted with diethyl
ether (50 mL × 3). The combined organic extracts were dried
with Mg2SO4, filtered, and concentrated at reduced pressure.
The crude materials were purified by silica gel column chro-
matography (2.8 × 10 cm, CHCl3 (500 mL), then CHCl3/MeOH
) 19/1, (800 mL)). The yield of 3 was 75% (664 mg). 1H NMR
(Me2SO-d6) δ 12.0 and 11.3 (each br s, each 1H, 1-NH and
2-NH), 7.31-6.72 (m, 13H, pheny of trityl), 6.28 (t, 1H, J ) 7.0
Hz, 1′-H), 5.24 (d, 1H, J ) 4.9 Hz, 3′-OH), 4.45 (m, 1H, 3′-H),
3.96 (m, 1H, 4′-H), 3.71 and 3.70 (each s, each 3H, O-CH3), 3.38-
3.08 (m, 2H, 5′-CH2), 2.99 (m, 1H, 2′-H), 2.73 (m, 1H, CH(CH3)2),
2.48 (s, 3H, 8-CH3), 2.20 (m, 1H, 2′-H), 1.12 and 1.11 (each d,
each 3H, J ) 6.8 Hz, C(CH3)2).
3′-O-[(2-Cya n oeth oxy)(d iisop r op yla m in o)p h osp h in o]-5′-
O-(4,4′-d im et h oxyt r it yl)-N2-isob u t yr yl-8-m et h yl-2′-d eox-
ygu a n osin e (4). N2- and 5′-O-diprotected 8-methyl-2′-deox-
yguanosine (3, 102 mg, 0.16 mmol) was dried over P2O5. After
compound 3 was coevaporated with dry CH2Cl2-benzene, CH2-
Cl2 (1 mL), triethylamine (54 µL, 0.388 mmol, 2.5 equiv mol),
and 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (52 µL,
0.233 mmol, 1.5 equiv mol) were added, and the mixture was
stirred at room temperature for 30 min. Formation of products
was monitored by TLC (silica gel, CHCl3/MeOH ) 19/1, Rf
values of products (R/S) were 0.42 and 0.51 and that of 3 was
0.19). After solvent was removed by evaporation, 5 mL of
tetrahydrofuran-benzene (1:4) was added and the mixture was
stirred for 10 min. The precipitate was removed by filtration
and evaporated to remove the solvent. Coevaporation of the
residue with dry benzene (5 mL × 2) gave 4 in 96% (128 mg).
Without further purification, the product was dried over P2O5
and used for subsequent oligodeoxynucleotide synthesis.
analyses were carried out using a Shimadzu LC10AD apparatus
equipped with a photodiode array UV detector SPD-M6A (Kyoto,
J apan). Oligodeoxynucleotides were synthesized by an Applied
Biosystems automated solid phase DNA synthesizer Model 392
(CA).
8-Meth yl-2′-d eoxygu a n osin e (1) (Sch em e 1). dG (2.0 g,
7.5 mmol) was added to an ice-cold solution of FeSO4‚7H2O (8.3
g, 30 mmol) in 400 mL of 1 N H2SO4 with stirring. tert-Butyl
hydroperoxide (80%, 2.5 g, 22 mmol) in 40 mL of H2O was added
dropwise to this solution during 4 min, and the mixture was
left standing for 1 min. Aqueous 1 N NaOH was then added to
the mixture to adjust neutral pH. For analysis of 8-MedG
formation, LiChrospher 100 RP-18(e) column (Merck, 4 × 125
mm, 5 µm) was eluted with H2O-MeOH (0-50% linear gradient
of MeOH over 30 min) at a flow rate of 1 mL/min. Retention
times (tR) of Gua, 8-MeGua, dG, and 8-MedG were 4.3, 7.6, 9.6,
and 12.7 min, respectively. Approximate yields of products were
as follows: 8-MedG (1) (59%), 8-MeGua (9%), Gua (10%), and
unreacted dG (22%). After solvent was removed by lyophiliza-
tion, products were extracted from the residue with hot MeOH
(200 mL × 2). 8-MedG was separated, using silica gel column
chromatography (4.4 × 30 cm, CHCl3 0.5 L, CHCl3/MeOH )
9/1, 2.5 L, and 17/3, 6 L), and further purified by Sephadex LH
20 column chromatography (2.5 × 30 cm, MeOH). After removal
of solvent, white powder was obtained in 29% yield (606 mg).
Recrystallization from MeOH gave analytically pure white
powder of 1. This is the first time that the mp (226-229 °C) of
Syn th esis of Oligodeoxyn u cleotides Con tain in g 8-MedG.
The following oligodeoxynucleotides containing 8-MedG were
prepared by the phosphoramidite method using an automated
solid phase DNA synthesizer: 5′CCTTCXCTACTTTCCTCTC-
1
8-MedG has been determined; H NMR (Me2SO-d6) δ 10.59 (s,
1H, 1-NH), 6.33 (s, 2H, 2-NH2), 6.13 (dd, 1H, J 1′,2′a ) 8.5 Hz,
J 1′,2′b ) 6.3 Hz, 1′-H), 5.25 (d, 1H, J 3′,3′-OH ) 4.2 Hz, 3′-OH), 4.98
(t, 1H, J 5′a,5′-OH ) J 5′b,5′-OH ) 5.6 Hz, 5′-OH), 4.34 (dddd, 1H, J 2′a,3′
) 6.6 Hz, J 2′b,3′ ) 2.7 Hz, J 3′,4′ ) 3.2 Hz, 3′-H), 3.77 (dt, 1H,
J 4′,5′a ) J 4′,5′b ) 4.9 Hz, 4′-H), 3.62-3.57 (ddd, 1H, J 5′a,5′b ) 11.7,
5′-Ha), 3.54-3.48 (ddd, 1H, 5′-Hb), 2.84-2.77 (ddd, 1H, J 2′a,2′b
) 13.9 Hz, 2′-Ha), 2.40 (s, 3H, 8-CH3), 2.08-2.02 (ddd, 1H, 2′-
Hb); UV λmax nm (pH 1) 257 (ꢀ ) 13 300), 277 (sh) (9700), (H2O)
251 (14 200), 274 (sh) (9100), (pH 12) 257 (12 500). Anal. Calcd
for C11H15N5O4: C, 42.86; H, 5.39; N, 22.72. Found: C, 42.68;
H, 5.27; N, 22.93. FAB-MS: m/z 282 (M + H)+, 166 (M - sugar
+ H)+.
5
CATTT (sequence 2 in Table 2) and ′GCGCCXGCGGTG, where
X is 8-MedG. Purification of oligodeoxynucleotides was carried
out using an OPC column (Applied Biosystems). The following
procedure was used for base analysis. A 20 µL aliquot of
nuclease P1 (1 mg/mL) was added to a solution of oligodeoxy-
nucleotides (1 OD260 unit) in 280 µL of 20 mM sodium acetate
buffer (pH 4.8) and incubated at 37 °C for 30 min. To this
solution, 200 µL of Tris-HCl buffer (pH 7.5) and alkaline
phosphatase (3.5 units/100 µL) were added, and the mixture
was incubated at 37 °C for another 30 min for 12-mer and 10
min for 24-mer. Composition of nucleosides was examined by
HPLC using an ODC column (4 × 250 mm). Elution was
performed with a linear gradient of MeOH in 0.67 M phosphate
buffer (pH 7.0) at a flow rate of 0.8 mL/min (0-50% MeOH with
an elution time of 120 min for 12-mer, and 0-5% MeOH with
an elution time of 5 min, then 5-100% MeOH with an elution
time of 115 min for 24-mer). The data of base composition for
each 8-MedG-modified oligodeoxynucleotide were consistent
with the calculated values (data not shown).
N2-Isob u t yr yl-8-m et h yl-2′-d eoxygu a n osin e (2). After
8-MedG (1, 500 mg, 1.77 mmol) was coevaporated with dry
pyridine (5 mL) twice, it was suspended in 18 mL of dry
pyridine. Chlorotrimethylsilane (1.12 mL, 8.85 mmol) was
added to this solution, and the mixture was left standing for 30
min in an ice-cold water bath. Isobutyric anhydride (1.48 mL,
8.85 mmol) was then added, and the mixture was left standing
at room temperature for 2 h. After the mixture was cooled again
in an ice-cold water bath, 3.7 mL of H2O was added, followed
by addition of 3.7 mL of concentrated NH4OH. After 30 min,
solvent was removed by evaporation. The residue was dissolved
in 15 mL of H2O and washed with diethyl ether (30 mL × 3).
Aqueous phase was reduced to two-thirds volume and left at 4
°C for 1 day. A white precipitate was collected in 77% yield
Exp er im en ts for Miscod in g Sp ecificity. Gen er a l. Organ-
ic chemicals used for the synthesis of oligodeoxynucleotides were
supplied by Aldrich Chemical (Milwaukee, WI). [γ-32P]ATP
(specific activity >6000 Ci/mmol) was obtained from Amersham
Corp. (Arlington Heights, IL). Cloned exo- Klenow fragment

1280 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Kohda et al.
Ta ble 1. P r od u ct An a lysis
of Escherichia coli DNA polymerase I (21 200 units/mg of
protein) was purchased from United States Biochemical Corp.
(Cleveland, OH), calf thymus DNA pol R (30 000 units/mg of
protein) was from Molecular Biology Resources, Inc. (Milwaukee,
WI), and T4 DNA ligase (400 units/µL) was from New England
BioLabs (Beverly, MA). HPLC grade acetonitrile, triethylamine,
and distilled water were purchased from Fisher Chemical
(Pittsburgh, PA). A Waters 990 HPLC instrument equipped
with a photodiode array detector (Milford, MA) was used for
the separation and purification of oligodeoxynucleotides.
b
%
conditiona
dG
8-MedG
Gua
8-MeGua
1 N H2SO4
0 °C, 5 min
0 °C, 7.5 min
0 °C, 10 min
25 °C, 5 min
0.1 N H2SO4
0 °C, 5 min
0 °C, 10 min
0 °C, 35 min
25 °C, 5 min
22.3
12.5
9.4
59.4
52.6
44.8
35.0
9.8
15.2
17.2
19.5
8.5
19.7
28.7
40.9
4.6
92.5
91.1
88.0
75.4
7.5
8.9
5.3
0
0
6.7
7.8
0
0
0
2.6
Syn th esis of Oligod eoxyn u cleotid es. Unmodified DNA
templates (sequences 1 and 2, X ) dG), primer, and standard
markers listed in Table 2 were prepared by the phosphoramidite
method using an automated DNA synthesizer (20). These
oligonucleotides and modified DNA template (sequence 2, X )
8-MedG) were purified on a Waters reverse-phase µBondapak
C18 (3.9 × 300 mm) using a linear gradient of 0.05 M triethyl-
ammonium acetate (pH 7.0) containing 10-20% acetonitrile
with an elution time of 60 min at a flow rate of 1.0 mL/min as
described previously (21). DNA templates and primers were
further purified by electrophoresis on 20% polyacrylamide gel
in the presence of 7 M urea (35 × 42 × 0.04 cm) (22). The
oligomers recovered from PAGE were again subjected to HPLC
to remove urea. Oligonucleotides were labeled at the 5′ termi-
nus by treating with T4 polynucleotide kinase in the presence
of [γ-32P]ATP (23) and subjected to electrophoresis to establish
homogeneity. The position of the oligomers was established by
autoradiography using Kodak Xomat XAR film.
P r im er Exten sion Rea ction s. Using a 32P-labeled 10-mer
(0.5 pmol, sequence 5 in Table 2) primed with an unmodified or
8-MedG-modified 24-mer template (1.0 pmol, sequence 2),
primer extension reactions catalyzed by exo- Klenow fragment
or pol R were carried out at 25 °C in 10 mL of a buffer containing
all four dNTPs (100 µM each) (19). The reaction buffer for exo-
Klenow fragment contained 50 mM Tris-HCl (pH 8.0), 8 mM
MgCl2, and 5 mM 2-mercaptoethanol. The buffer for pol R
contained 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 2 mM
dithiothreitol, and BSA (0.5 µg/µL). Reactions were stopped by
adding formamide dye and heating to 95 °C for 3 min. Samples
were subjected to electrophoresis on a 20% polyacrylamide gel
containing 7 M urea (35 × 42 × 0.04 cm). Bands were identified
by autoradiography and excised from the gel, and the radioac-
tivities were measured by a Wallac liquid scintillation counter.
14.2
a
To the solution of dG (20 mg) and FeSO4‚7H2O (83 mg) in 4
mL of 1 or 0.1 N H2SO4 with stirring was added dropwise a
solution of tert-butyl hydroperoxide (80%, 25 mg) in 0.4 mL of H2O
during 5 min at 0 or 25 °C. The mixture was then left standing
for 0-30 min at the same temperature. At the indicated time (5
min for addition plus standing time), an aliquot was taken and
neutralized with aqueous 1 N NaOH. The formation of products
b
formed was analyzed by HPLC. Yield was expressed as mol %.
were dissolved in a 10 mM sodium cacodylate buffer (pH 7.0)
containing 100 mM NaCl and 1 mM EDTA, heated at 80 °C for
10 min with complementary strand where the nucleotide
opposite 8-MedG was dC, dA, dT, or dG, and then cooled
gradually to room temperature for the annealing. Thermal
denaturation curves were recorded at 260 nm on a UV spec-
trometer with a temperature controller, and the melting tem-
perature was evaluated. van’t Hoff transition enthalpies (∆H°),
entropies (∆S°), and free energies (∆G°) were determined by
calculations based on the slope and intercept of a 1/Tm versus
ln(Ct/4) plot of the following equations (25):
1/Tm ) (R/∆H°) ln(Ct/4) + ∆S°/∆H°
∆G° ) ∆H° - T∆S°
where Ct is the total concentration of single strands.
Resu lts
Syn th esis of 8-Med G Der iva tives. 8-Methylgua-
nosine was reported to be formed directly from Gua by
radical methylation using the tert-butyl hydroperoxide-
FeSO4 system in 1 N H2SO4 at room temperature for
about 30 min (11). When the same reaction conditions
were used to prepare 8-MedG, the glycosidic bond of
8-MedG was readily cleaved (data not shown). To obtain
optimum conditions for the synthesis of 8-MedG, the
formation of 8-MedG was measured under various reac-
tion conditions using HPLC (Table 1). When 1 N H2SO4
was used, the maximum yield (60%) of 8-MedG was
observed at 0 °C for 5 min incubation. Longer reaction
times and higher reaction temperatures decreased the
yield of 8-MedG. Weakly acidic reaction conditions also
decreased the amount of 8-MedG formation. Purification
using column chromatography resulted in a tolerable
yield of 8-MedG (29%).
Qu a n tita tion of Miscod in g Sp ecificity. One microgram
of 8-MedG-modified 24-mer (sequence 2 in Table 2) was phos-
phorylated at the 5′ terminus using 7 µL of T4 kinase and 10
mM ATP (23) and then ligated to a 14-mer (1.2 µg, sequence 3)
at 8 °C overnight using 3 µL of T4 DNA ligase, 2 µL of 10 mM
ATP, and an 18-mer template (1.5 µg, sequence 4). The
resultant 38-mer (sequence 1) was isolated by HPLC as de-
scribed above (21) and used as a DNA template. Using a 38-
mer template (1.0 pmol) primed with a 32P-labeled 12-mer (0.5
pmol, sequence 6), primer extension reactions were conducted
at 30 °C for 1 h in the presence of four dNTPs, using 0.05 unit
of exo- Klenow fragment or 2.4 unit of pol R as described above.
The molar ratios of the exo- Klenow fragment:template and of
pol R:template were estimated to be approximately 1:29 and 1:2,
respectively. The reaction samples were subjected to 20% PAGE
containing 7 M urea (35 × 42 × 0.04 cm). The fully-extended
products were recovered from the gel, annealed with an un-
modified 38-mer (sequence 1), and incubated with EcoRI restric-
tion enzyme (100 units) at 30 °C for 1 h and subsequently at 15
°C for 1 h to cleave completely as shown in Figure 2. To quantify
all base substitutions and deletions, the samples were subjected
to electrophoresis on two-phase 20% polyacrylamide gels (15 ×
72 × 0.04 cm) containing 7 M urea in the upper phase and no
urea in the lower phase (17).
P r ep a r a tion of Oligom er Con ta in in g 8-Med G.
8-MedG (1) was isobutyrylated at the 2-NH2 group to give
a 77% yield of N2-isobutyryl-8-methyl-2′-deoxyguanosine
(2), and the subsequent dimethoxytritylation at the 5′-
O-position gave 5′-O-(4,4′-dimethoxytrityl)-N2-isobutyryl-
8-methyl-2′-deoxyguanosine (3) (75%). The phosphora-
midite of compound 3 (4) was prepared by the established
protocol (26). Compound 4 was used for the synthesis of
the following 8-MedG-modified oligomers by solid phase
DNA synthesizer: 5′CCTTCXCTACTTTCCTCTCCATTT
Mea su r em en t of Tm . Melting temperature (Tm) of oligode-
oxynucleotide duplex containing 8-MedG was measured under
the three different concentrations (1.1, 2.2, and 3.3 µM), using
12-mer oligodeoxynucleotide (Table 3). The sequence of the 12-
mer was selected from the c-Ha-ras gene (24); 8-MedG is
inserted in the first position of codon 12. Oligodeoxynucleotides
5
(sequence 2 in Table 2) and ′GCGCCXGCGGTG, where
X is 8-MedG. The 24-mer was used for the experiment

8-Methyl-dG DNA Adduct
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1281
Ta ble 2. Sequ en ce of Oligod eoxyn u cleotid esa
a
Sequence of templates, primers, and standard markers. X )
8-MedG or dG; N ) dC, dA, dG, or dT.
F igu r e 2. Diagram of the primer extension methods and
analysis of reaction products.
unmodified template was similar to that of the 8-MedG
template (Figure 1C). In contrast, when pol R, a mam-
malian replicative enzyme, was used, the primer exten-
sion reactions were retarded at the position of the
8-MedG lesion (Figure 1B); a portion of the primers
passed the lesion forming the fully-extended products.
When 0.6 and 1.2 units of pol R were used, the amounts
of fully-extended products formed on the 8-MedG-modi-
fied template were 14 and 6.8 times lower, respectively,
than that of unmodified template (Figure 1D).
When a large amount of enzyme was used for the
primer extension reaction, a blunt-end addition reaction
(27, 28) occurred at the 3′ terminus, as observed in Figure
1A,B (lanes 5, 13, and 16). The formation of blunt-end
products failed to quantify base substitutions and dele-
tions (17). Using a 38-mer template (sequence 1 in Table
2) instead of 24-mer (sequence 2), fully-extended reaction
products were cleaved with EcoRI restriction enzyme and
subjected to a two-phase gel electrophoresis for quantify-
ing the miscoding specificity of 8-MedG (Figure 2). The
standard mixture of six 32P-labeled oligodeoxynucleotides
containing dC, dA, dG, or dT opposite the lesion and one-
or two-base deletion are completely resolved by this
method (Figure 3A, lanes 1 and 4). When the exo-
Klenow fragment (lane 2) or pol R (data not shown) was
used, DNA synthesis on an unmodified template leads
to the expected incorporation of dCMP opposite dG.
Using the exo- Klenow fragment, dCMP (77.0%) was
preferentially incorporated opposite 8-MedG, along with
the incorporation of dGMP (1.10%), dAMP (0.41%), and
one- (0.38%) and two-base (0.81%) deletions (lane 3).
When pol R was used, the incorporation of dCMP (78.3%)
opposite 8-MedG was also detected predominantly with
small amounts of incorporation of dGMP (0.82%) and
dAMP (0.38%) (lane 5).
F igu r e 1. Primer extension reactions catalyzed by DNA
polymerases on 8-MedG-modified template. Using an unmodi-
fied or 8-MedG-modified 24-mer template (sequence 2 in Table
2) primed with a 32P-labeled 10-mer (sequence 5), primer
extension reactions were conducted at 25 °C for 1 h using a
variable amount of the exo- Klenow fragment (A) or pol R (B)
as described under Experimental Procedures. One-third of the
reaction mixture was subjected to denaturing 20% polyacryla-
mide gel electrophoresis (35 × 42 × 0.04 cm). The bands of fully-
extended products observed in (A) and (B) were cut from PAGE,
and the radioactivities were measured by a liquid scintillation
counter. Exo- Klenow fragment (C) and pol R (D).
Th er m od yn a m ic Sta bility of Ba se P a ir s Con ta in -
in g 8-Med G. An unmodified or 8-MedG-modified 12-
mer was annealed with complementary strands (Table
3). The melting temperatures (Tms) were measured
under the three different concentrations (1.1, 2.2, and 3.3
µM). Plots of 1/Tm versus ln(Ct/4) showed a straight line
(data not shown), and the thermodynamic parameters
(∆H°, ∆S°, and ∆G°) were calculated using the equation
reported previously (25). The Tms and thermodynamic
parameters obtained are summarized in Table 3. The
correct base pair, dG:dC, showed the highest Tm (67.3
°C). The 8-MedG:dC showed a higher Tm than that of
the other pairs with 8-MedG. The Tm of 8-MedG:dG was
2.5 °C higher than that of the mismatched dG:dG pair.
of the miscoding specificity, and the 12-mer was for the
measurements of Tm.
Miscod in g Sp ecificity of Oligod eoxyn u cleotid e
Con ta in in g 8-Med G. Primer extension reactions cata-
lyzed by the exo- Klenow fragment of E. coli DNA
polymerase I were conducted in the presence of four
dNTPs (Figure 1A). Using an unmodified template
(sequence 2, X ) dG in Table 2), primer extension
occurred smoothly to form the fully-extended products.
On the 8-MedG-modified template (sequence 2, X )
8-MedG), the primer extension was not blocked at the
lesion and readily formed the fully-extended products.
The amount of fully-extended products formed on an

1282 Chem. Res. Toxicol., Vol. 9, No. 8, 1996
Kohda et al.
synthesis of oligodeoxynucleotides containing 8-MedG,
where 3′,5′-di-O-acetyl-2′-deoxyguanosine (31) or N2-
isobutyryl-2′-deoxyguanosine (32) was used as a sub-
strate for radical methylation.
Primer extension reactions catalyzed by the exo-
Klenow fragment of E. coli DNA polymerase I or mam-
malian pol R were conducted in the presence of all four
dNTPs. When the exo- Klenow fragment was used, the
primer was extended readily past 8-MedG, forming fully-
extended products, similar to that observed with the
unmodified template (Figure 1A,C). However, primer
extension by pol R was retarded at the position opposite
8-MedG (Figure 1B,D). These results indicate that the
extension process past 8-MedG is different depending on
the DNA polymerase used.
Using a modified in vitro experimental system (Figure
2), base substitutions and deletions induced by 8-MedG
were quantified (17). Both exo- Klenow fragment and
pol R incorporated predominantly dCMP, the correct
base, opposite 8-MedG. In addition, small amounts of
incorporation of dGMP (0.82-1.10%) and dAMP (0.38-
0.41%) opposite the lesion were detected (Figure 3A). The
polymerase activity of DNA polymerase III, an E. coli
replicative enzyme, is carried out by the R subunit (33).
When the R subunit of pol III was used, dCMP only was
incorporated opposite 8-MedG (data not shown). Using
a similar experimental condition with 0.05 unit of the
exo- Klenow fragment, one of the authors (S.S.) reported
that O6-methyl-2′-deoxyguanosine (O6MedG) directed
incorporation of dTMP (75.5%), along with small amounts
of incorporation of dCMP (5.27%) (17). When pol R (2.5
units) was used, incorporation of dTMP (24.2%) and
dCMP (4.78%) opposite O6MedG was observed (17).
Thus, the miscoding specificity of 8-MedG differs from
that of O6MedG. The miscoding frequencies of 8-MedG
were 25-50 times less than that of O6MedG. Since the
amount of 8-MedG induced by 1,2-dimethylhydrazine in
tissue DNA of rats was similar to that of O6MeG (13),
8-MedG may be weakly mutagenic predicting the genera-
tion of G f C and G f T transversions in cells.
Two-base deletion was observed opposite 8-MedG when
the exo- Klenow fragment was used (Figure 3A). Based
on studies with several DNA adducts, a general mecha-
nism of frameshift deletion was proposed (18). When the
extension of the newly inserted dNMP opposite the lesion
is blocked, the newly inserted dNMP and/or its 5′
flanking bases in the primer could pair with bases 5′ to
the lesion in the template to form deletions (18). Since
a small amount of dAMP was inserted opposite 8-MedG
under the reaction conditions containing a single dNTP
(data not shown), the newly inserted dAMP and its 5′
flanking G could pair with C 5′ to the lesion in the
template to form two-base deletion (Figure 3B).
F igu r e 3. Quantitation of miscoding specificities. (A) Using
a 38-mer template (sequence 1 in Table 2) primed with a 5′-
32P-labeled 12-mer (sequence 6), primer extension reactions were
conducted for 1 h at 30 °C, using 0.05 unit of exo- Klenow
fragment for the unmodified and 8-MedG-modified templates
and 2.4 unit of pol R for 8-MedG-modified template. The fully-
extended products recovered from PAGE were cleaved by EcoRI
restriction enzyme at 30 °C for 1 h and subsequently at 15 °C
for 1 h as described under Experimental Procedures. The
reaction samples were subjected to a two-phase 20% polyacry-
lamide gel electrophoresis (15 × 72 × 0.04 cm). Mobilities of
reaction products were compared with those of 18-mer standards
(sequences 7-9) containing dC, dA, dG, or dT opposite the lesion
and one-base (∆1) or two-base (∆2) deletions (lanes 1 and 4). (B)
Proposed mechanism for two-base deletion.
Ta ble 3. Meltin g Tem p er a tu r es (Tm ) a n d
Th er m od yn a m ic P a r a m eter s for Mod ified a n d
Un m od ified Oligod eoxyn u cleotid e Du p lexes
b
base
paira
Tm
∆H°
(kcal/mol)
∆S°
(cal/Kmol)
∆G°298
(kcal/mol)
(°C)
GC
GA
GT
GG
67.3
60.4
58.5
55.4
-110.4
-58.4
-66.3
-68.5
-299
-149
-174
-182
-21.4
-14.1
-14.5
-14.3
GC
GA
GT
GG
61.8
55.6
53.1
57.9
-90.3
-86.4
-79.5
-86.4
-244
-236
-218
-234
-17.8
-16.1
-14.6
-16.6
a
b
G ) 8-MedG. The concentration of duplexes was 3.3 µM.
However, the other pairs with 8-MedG showed lower Tms
than the corresponding pairs with unmodified dG. Al-
though the ∆G° value of 8-MedG:dC was lower than that
of the other pairs with 8-MedG, the ∆G° values of
8-MedG:dG and 8-MedG:dA were 2.3 and 2.0 kcal/mol
lower than that of mismatched dG:dG and dG:dA,
respectively.
Methyl radicals and hydroxy radicals attack at the C8-
position of dG in duplex DNA to create 8-MedG (12, 34)
and 7,8-dihydro-8-oxodG (35, 36), respectively. 7,8-
Dihydro-8-oxodG is a mutagenic lesion (37-40) and
promotes the incorporation of dCMP and dAMP opposite
the lesion during DNA synthesis; the ratio of dCMP and
dAMP varies depending on the DNA polymerase used
(19). Since this adduct exists predominantly as the 6,8-
diketo form (8-oxodG) under physiological conditions (41,
42), 8-oxodG could be in the anti conformation to pair
with dCMP, forming a Watson-Crick base pair (43);
alternatively, 8-oxodG could be in the syn conformation
to pair with dAMP, forming a Hoogsteen base pair (44).
However, since 8-MedG can not exist as the other
Discu ssion
8-MedG was reported to be prepared from 8-methyl-
guanine by enzymatic deoxyribosylation (29, 30), but the
yield was too low to characterize it unequivocally. We
have prepared 8-MedG directly from dG using a modifi-
cation of the radical methylation system (11). The
phosphoramidite of the N2- and 5′-O-diprotected 8-MedG
was used for the synthesis of oligodeoxynucleotides
containing 8-MedG using a solid phase DNA synthesizer.
These results are consistent with a recently reported

8-Methyl-dG DNA Adduct
Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1283
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tautomers, the miscoding specificities of 8-MedG may
differ from that of 8-oxodG and the miscoding frequencies
of 8-MedG may be less than that of 8-oxodG.
The miscoding properties of 8-MedG are quite similar
to that of N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-
C8-AF), which permitted incorporation of dCMP only in
reactions catalyzed by exo- and exo+ Klenow fragment
(17, 18, 45) and promoted predominant incorporation of
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8-MedG can also be in the syn conformation in duplex
oligomers (32), the syn conformation of 8-MedG may pair
with dAMP or dGMP, as proposed in Figure 4.
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The pair of 8-MedG:dG showed higher Tm and lower
∆G° values than the corresponding dG:dG (Table 3).
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dG:dA, the ∆G° value of 8-MedG:dA was lower than that
of dG:dA. These data indicate that 8-MedG:dA and
8-MedG:dG are thermodynamically more stable than the
corresponding mismatched base pairs. Since the Tm of
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8-MedG:dC was the lowest among the duplexes contain-
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more stable than the other pairs with 8-MedG. Although
the sequence context of oligomer used for thermodynamic
study was different from that used for the in vitro
mutagenesis study, the ∆G° values inversely parallel the
frequency of base incorporation opposite 8-MedG as
follows: dCMP > dGMP > dAMP . dTMP. Thus, the
thermodynamic stabilities may correlate to the miscoding
specificities induced by 8-MedG.
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