Comparison of the in vitro replication of the 7-(2-Oxoheptyl)-1, N 2-etheno-2′-deoxyguanosine and 1, N 2-etheno- 2′-deoxyguanosine lesions by sulfolobus solfataricus P2 DNA polymerase IV (Dpo4)

Article abstract of DOI:10.1021/tx100082e

Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno- dGuo adduct, which is derived from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-fam

Full text of DOI:10.1021/tx100082e

1330
Chem. Res. Toxicol. 2010, 23, 1330–1341
Comparison of the in Vitro Replication of the
7-(2-Oxoheptyl)-1,N2-etheno-2′-deoxyguanosine and
1,N2-Etheno-2′-deoxyguanosine Lesions by Sulfolobus solfataricus P2
DNA Polymerase IV (Dpo4)
Plamen P. Christov,† Katya V. Petrova,† Ganesh Shanmugam,† Ivan D. Kozekov,†
Albena Kozekova,† F. Peter Guengerich,‡,§ Michael P. Stone,†,‡,§ and
Carmelo J. Rizzo*,†,‡,§
Department of Chemistry, Department of Biochemistry, and Center in Molecular Toxicology,
Vanderbilt UniVersity, NashVille, Tennessee 37235-1822
ReceiVed March 3, 2010
Oligonucleotides were synthesized containing the 7-(2-oxoheptyl)-etheno-dGuo adduct, which is derived
from the reaction of dGuo and the lipid peroxidation product 4-oxo-2-nonenal. The in vitro replication
of 7-(2-oxoheptyl)-etheno-dGuo by the model Y-family polymerase Sulfolobus solfataricus P2 DNA
Polymerase IV (Dpo4) was examined in two sequences. The extension products were sequenced using
an improved LC-ESI-MS/MS protocol developed in our laboratories, and the results were compared to
that of the 1,N2-etheno-dGuo adduct in the same sequence contexts. Both etheno adducts were highly
miscoding when situated in 5′-TXG-3′ local sequence contexts with <4% of the extension products
being derived from error-free bypass. The major extension products resulted from the misinsertion of
Ade opposite the adduct and a one-base deletion. The major extension products from replication of the
etheno lesions in a 5′-CXG-3′ local sequence context were the result of misinsertion of Ade, a one-base
deletion, and error-free bypass. Other minor extension products were also identified. The 7-(2-oxoheptyl)-
etheno-dGuo lesion resulted in a larger frequency of misinsertion of Ade, whereas the 1,N2-etheno-dGuo
gave more of the one-base deletion product. Conformational studies of duplex DNA containing the 7-(2-
oxoheptyl)-etheno-dGuo in a 5′-TXG-3′ sequence context by NMR indicated the presence of a pH-
dependent conformational transition, likely involving the glycosyl bond at the adducted guanosine; the
pKa for this transition was lower than that observed for the 1,N2-ε-dGuo lesion. However, the 7-(2-
oxoheptyl)-etheno-dGuo lesion, the complementary Cyt, and both flanking base pairs remained disordered
at all pH values, which is attributed to the presence of the hydrophobic heptyl group of the 7-(2-oxoheptyl)-
etheno-dGuo lesion. The altered pKa value and the structural disorder at the 7-(2-oxoheptyl)-etheno-
dGuo lesion site, as compared to the same sequence containing the 1,N2-etheno-dGuo, may contribute to
higher frequency of misinsertion of Ade.
adducts are uniquely derived from oxidative stress. 7-(2-
Oxoheptyl)-ε-dGuo and 3-(2-oxoheptyl)-ε-dCyd were detected
from the small intestines of min mice, which are a colorectal
cancer mouse model that overexpresses cyclooxygenase-2 (13).
Structurally related substituted etheno adducts have been
characterized from 4-oxo-2-hexenal (14-17), a peroxidation
product from ω3-fatty acids, buten-1,4-dial (18-21), which is
derived from the metabolism of furan (22), and 4-oxo-2-
pentenal, which is derived from the metabolism of N-nitros-
opyrrolidine (23-25), methylfuran (26), and the ꢀ,δ-elimination
of an abasic site in DNA (27-29). The N2,3-ε-dGuo adduct
Introduction
Etheno (ε) adducts of dGuo, dAdo, and dCyd result from
the reaction of DNA with epoxides of vinyl monomers and have
been implicated, at least in part, in the hepatocarinogenicity of
vinyl chloride (1, 2). Subsequent studies identified background
levels of etheno adducts in unexposed test animals (3). Lipid
peroxidation products are believed to be the source of the
endogenous etheno adducts (4-6). In a series of studies, Blair
and co-workers characterized the substituted etheno adducts
7-(2-oxoheptyl)-ε-dGuo1 (1) (7), 7-(2-oxoheptyl)-ε-dAdo (2)
(8, 9), 3-(2-oxoheptyl)-ε-dCyd (3) (10, 11), and 7-(2-oxoheptyl)-
ε-Guo (12) from the reaction of the lipid peroxidation product
4-oxo-2-nonenal (ONE) with the appropriate nucleoside or
nucleic acid (Figure 1); the 2-oxoheptyl-substituted etheno
1 Abbreviations: 7-(2-oxoheptyl)-ε-dGuo, 3-(2-deoxy-ꢀ-D-erythro-pento-
furanosyl)-2,4-dihydro-7-(2-oxoheptyl)-9H-imidazo[1,2-a]purin-9-one; 7-(2-
oxoheptyl)-ε-dAdo, 3-(2-deoxy-ꢀ-D-erythro-pentofuranosyl)-3-(2-oxoheptyl)-
3H-imidazo[2,1-i]-purine; 3-(2-oxoheptyl)-ε-dCyd, 6-(2-deoxy-ꢀ-D-erythro-
pentofuranosyl)-3-(2-oxoheptyl)-imidazo[1,2-c]pyrimidin-5(6H)-one; 7-(2-
oxoheptyl)-ε-Guo, 3-(ꢀ-D-erythro-pentofuranosyl)-2,4-dihydro-7-(2-oxoheptyl)-
9H-imidazo[1,2-a]purin-9-one; ε, etheno; M1dGuo, 3-(2-deoxy-ꢀ-D-erythro-
pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one; ONE, 4-oxo-2-nonenal;
UDG, uracil DNA glycosylase; DMTr, dimethoxytrityl; pol, DNA poly-
merase; CID, collision induced dissociation; UPLC, ultraperformance liquid
chromatography; ESI, electrospray ionization; MALDI-TOF-MS, matrix
assisted laser desorption ionization time-of-flight mass spectrometry; Tm,
thermal melting temperature; NOE, nuclear Overhauser effect; NOESY,
nuclear Overhauser enhancement spectroscopy.
* To whom correspondence should be addressed. Department of
Chemistry, Vanderbilt University, VU Station B 351822, Nashville, TN
37235-1822. Tel: 615-322-6100. Fax: 615-343-1234. E-mail: c.j.rizzo@
vanderbilt.edu.
† Department of Chemistry.
‡ Department of Biochemistry.
§ Center in Molecular Toxicology.
10.1021/tx100082e  2010 American Chemical Society
Published on Web 06/28/2010

Bypass of 7-(2-Oxoheptyl)-ε-dGuo by Dpo4
Chem. Res. Toxicol., Vol. 23, No. 8, 2010 1331
described by our laboratories (36); the distribution of products
from the translesion synthesis of 7-(2-oxoheptyl)-ε-dGuo was
similar to that of the of 1,N2-ε-dGuo lesion. NMR studies of
the 7-(2-oxoheptyl)-ε-dGuo in the 5′-TXG-3′ sequence indicated
that the lesion underwent a pH-dependent conformational
transition, similar to the 1,N2-ε-dGuo lesion, but with a more
acidic pKa. The nature of this conformational transition remains
to be determined because the complementary Cyt and both
flanking base pairs remained disordered at all pH values
examined but likely involves rotation about the glycosyl bond
as observed for the 1,N2-ε-dGuo lesion (37, 38).
Experimental Procedures
7-(2-Oxoheptyl)-1,N2-etheno-2′-deoxyguanosine (1). dGuo·H2O
(114 mg, 0.40 mmol) and K2CO3 (100.0 mg, 0.72 mmol) were
suspended in DMF (5 mL) and stirred at room temperature for 15
min. A solution of ONE (39) (125 mg, 0.80 mmol) in DMF (5
mL) was prepared; a portion of this solution (2.5 mL, 0.40 mmol)
was added to the reaction, and a second portion (2.5 mL, 0.40
mmol) was added after 60 min. The reaction was stirred for an
additional 6 h with the progress of the reaction monitored by HPLC
(gradient B). The reaction was poured over crushed ice (50 mL).
After neutralization with 1% HCl (w/v), the mixture was allowed
to stand at room temperature overnight. The precipitated solid was
removed by centrifugation, rinsed with water, frozen on dry ice,
and dried on a lyophilizer to afford 1 (61.0 mg). The filtrate was
concentrated in vacuo, and the resulting solid isolated just as
described afforded additional 1 (27.0 mg). The purity of 1 (88.0
mg, 55% yield) was judged to be >95% by HPLC (gradient B)
and used without further purification. 1H NMR (600 MHz, DMSO-
d6) δ 12.22 (br s, 1H, NH), 8.06 (s, 1H, H-2), 7.13 (s, 1H, H-6),
6.20 (dd, 1H, J ) 7.2, 0.9 Hz, H-1′), 5.28 (d, 1H, J ) 2.7 Hz,
3′-OH), 4.95 (s, 1H, 5′-OH), 4.36 (m, 1H, H-3′), 4.10 (ABq, 2H,
J ) 12.2 Hz, ∆νAB ) 8.5 Hz, H-10), 3.83 (m, 1H, H-4′), 3.57 (m,
1H, H-5′), 3.51 (m, 1H, H-5′′), 2.59 (m, 1H, H-2′), 2.54 (m, 2H,
H-12), 2.23 (m, 1H, H-2′′), 1.50 (m, 2H, H-13), 1.25 (m, 4H, H-14,
H-15), 0.85 (t, 3H, J ) 7.1 Hz, H-16). 13C NMR (150 MHz, DMSO-
d6) δ: 205.6, 153.6, 149.7, 146.7, 136.8, 117.8, 115.9, 115.4, 87.6,
82.9, 70.7, 61.7, 41.1, 39.5, 39.4, 30.8, 22.7, 22.0, 13.8.
Figure 1. Structures of the nucleoside adducts of 4-oxo-2-nonenal
(1-3).
has been identified from the reaction of dGuo with chloro-
acetaldehyde and chlorooxirane (30, 31), but the analogous
etheno adduct from ONE and related 4-oxo-alkenal have not
been characterized to date.
The 3-(2-oxoheptyl)-ε-dCyd (3) adduct has been site-specif-
ically incorporated into a 13-mer oligonucleotide using the
corresponding phosphoramidite reagent (32). The modified
oligonucleotide was then ligated into a shuttle vector, and its
mutagenicity was evaluated in Escherichia coli and three human
cell lines (XPA, XPV, and repair-proficient GM637 cells). The
3-(2-oxoheptyl)-ε-dCyd adduct was found to be a strong block
to replication in all cell lines examined; however, the adduct
was highly miscoding when bypassed. In bacteria, the miscoding
frequency was up to 48% and consisted mostly of CfG
transversions. The miscoding frequency was >90% in the human
cell lines, with CfA transversions as the major mutation
followed by CfT transitions. Similar results were observed
when the parent unsubstituted ε-dCyd adduct was replicated in
simian kidney cells (33). The marked differences in the
miscoding properties of 3-(2-oxoheptyl)-ε-dCyd between the
human and bacterial cells were attributed to the different DNA
polymerases involved in translesion synthesis (32). The observa-
tion that the mutational spectrum and frequency was nearly
identical for wild type GM637 and pol η-deficient XPV cells
suggests a limited role for pol η in the bypass of 3-(2-oxoheptyl)-
ε-dCyd. Replication of the 3-(2-oxoheptyl)-ε-dCyd adduct in
mouse fibroblast cells in which the genes encoding pol η, ι, or
κ were individually knocked out did not qualitatively effect
translesion synthesis (34). Interestingly, insertion of dAde was
abolished when the lesion was replicated in cells in which the
ReV3 or ReV1 genes were inactivated, but dThy insertion was
not affected. These studies suggested that pol ꢁ/REV1 was
involved in mutagenic bypass of the 3-(2-oxoheptyl)-ε-dCyd
adduct leading to CfT transitions, while bypass by Y-family
polymerases are responsible for CfA transversions.
FAB HRMS+ m/z calcd for C19H26N5O5 [M + H]+, 404.1934;
found, 404.1917.
5,5′-N,O-Bis(4,4′-dimethoxytrityl)-7-(2-oxoheptyl)-1,N2-etheno-
2′-deoxyguanosine (4). Nucleoside 1 (65 mg, 0.16 mmol) was dried
by dissolving in anhydrous pyridine (5 mL) and evaporating the
solvent in vacuo on a rotary evaporator; the procedure was repeated
two additional times. To a stirred solution of 1 in anhydrous pyridine
(3 mL) were added 4,4′-dimethoxytrityl chloride (164 mg, 0.48
mmol) and diisopropylethylamine (85 µL, 62 mg, 0.48 mmol). The
reaction mixture was stirred for 5 h at room temperature under an
argon atmosphere. The solvent was removed in vacuo, and the
residue was purified by flash column chromatography on silica gel,
eluting with a gradient of CH2Cl2/pyridine (98:2, v/v) to CH2Cl2/
CH3OH/pyridine (98:1:1) to afford bis-tritylated nucleoside 4 (107
1
mg, 66%). H NMR (400 MHz, DMSO-d6) δ 7.78 (s, 1H, H-2),
7.32 (m, 2H, aromatic), 7.31-7.04 (m, 18H, aromatic), 6.94 (s,
1H, H-6), 6.86-6.81 (m, 8H, aromatic), 5.64 (t, 1H, J ) 6.2 Hz,
H-1′), 5.09 (d, 1H, J ) 4.9 Hz, 3′-OH), 4.14 (s, 2H, H-10), 3.82
(m, 2H, H-3′, H-4′), 3.71 and 3.68 (s, 12H, 4OCH3), 3.02 (m, 2H,
H-5′, H-5′′), 2.5 (m, 2H, H-12), 2.10 (m, 1H, H-2′), 1.78 (m, 1H,
H-2′′), 1.48 (m, 2H, H-13), 1.25 (m, 4H, H-14, H-15), 0.84 (t, 3H,
J ) 6.8 Hz, H-16).
We report here the site-specific synthesis of oligonucleotides
containing the 7-(2-oxoheptyl)-ε-dGuo lesion and the in vitro
replication of this lesion by the model Y-family polymerase
Dpo4 in two sequences. As we previously reported with 1,N2-
ε-dGuo in the same sequence contexts, the misincorporation
profile was dependent on the identity of the 5′-neighboring base
to the lesion (35). The products of translesion synthesis were
sequenced by an improved LC-ESI-MS-MS method previously
TOF ES MS+ m/z calcd for C61H62N5O9 [M + H]+, 1008.4548;
found, 1008.4548.
5′-O-(4,4′-Dimethoxytrityl)-7-(2-oxoheptyl)-1,N2-etheno-2′-
deoxyguanosine (5). A solution of 4 (107 mg, 0.11 mmol) in
CH3OH (10 mL) was heated at reflux for 15 min after which time
the solvent was removed in vacuo. The residue was purified by
flash column chromatography on silica gel, eluting with a gradient
of CH2Cl2/CH3OH/pyridine (95:3:2 to 94:4:2, v/v/v) to afford 5′-

1332 Chem. Res. Toxicol., Vol. 23, No. 8, 2010
ChristoV et al.
O-tritylated nucleoside 5 (67 mg, 90%). 1H NMR (400 MHz,
DMSO-d6) δ 7.93 (s, 1H, H-2), 7.32 (m, 2H, aromatic), 7.19 (m,
7H, aromatic), 7.13 (s, 1H, H-6), 6.78 (m, 4H, aromatic), 6.24 (t,
1H, J ) 6.4 Hz, H-1′), 5.32 (d, 1H, J ) 4.6 Hz, 3′-OH), 4.37 (m,
1H, H-3′), 4.12 (s, 2H, H-10), 3.94 (m, 1H, H-4′), 3.70 and 3.69
(s, 6H, 2OCH3), 3.19 (m, 1H, H-5′), 3.11 (m, 1H, H-5′′), 2.68 (m,
1H, H-2′), 2.55 (t, 2H, J ) 7.4 Hz, H-12), 2.31 (m, 1H, H-2′′), 1.5
(m, 2H, H-13), 1.32 (m, 4H, H-14, H-15), 0.86 (t, 3H, J ) 6.9 Hz,
H-16). 13C NMR (150 MHz, DMSO-d6) δ: 206.9, 167.5, 158.7,
154.2, 147.1, 144.6, 135.5, 135.4, 130.8, 129.0, 128.7, 128.0, 127.7,
127.0, 126.8, 118.3, 115.7, 113.0, 86.4, 86.4, 86.3, 83.1, 72.0, 68.0,
64.1, 55.1, 42.2, 39.7, 38.8,38.7, 31.3, 30.3, 28.9, 23.7, 23.2, 23.0,
22.5, 13.8, 13.7, 10.7.
5′-CGC ATX GAA TCC-3′ (9). Solid-phase synthesis on
1-µmol scale afforded 20 A260 units (158 nmols). MALDI-TOF-
MS (HPA) m/z calcd for [M - H]- 3766.6; found, 3766.5.
HPLC Separations. The purification of the oligonucleotides and
the analyses of reaction mixtures were performed on a Beckman
HPLC system (32 Karat software version 7.0, pump module 125)
with a diode array UV detector (module 168) monitoring at 260
nm using a Waters YMC ODS-AQ columns (250 mm ×4.6 mm,
1.5 mL/min for analysis and 250 mm ×10 mm, 5 mL/min for
purification). The following solvent gradients (v/v) were used.
Gradient A (CH3CN/0.1 M NH4 HCO2). Initially, 1% CH3CN;
a linear gradient from 1 to 8% CH3CN over 5 min; a linear gradient
from 8-12% CH3CN over 15 min; a linear gradient from 12-80%
CH3CN over 2 min; isocratic at 80% CH3CN for 1 min; then a
linear gradient to 1% CH3CN over 2 min.
FAB HRMS+ m/z calcd for C40H44N5O7 [M + H]+, 705.3241;
found, 705.3273.
Gradient B (CH3CN/H2O). Initially, 10% CH3CN; a linear
gradient to 80% CH3CN over 25 min; then a linear gradient to the
initial conditions over 5 min.
3′-O-[N,N-Diisopropylamino)-2-cyanoethoxyphosphinyl]-5′-O-
(4,4′-dimethoxytrityl)-7-(2-oxoheptyl)-1,N2-etheno-2′-deoxygua-
nosine (6). Nucleoside 5 (82 mg, 0.12 mmol) was evaporated from
anhydrous pyridine (3 × 5 mL) and dried overnight under vacuum.
The oily residue was dissolved in freshly distilled CH2Cl2 (25 mL),
and a 0.45 M solution of 1H-tetrazole in CH3CN (330 µL, 11 mg,
0.15 mmol) and 2-cyanoethyl-N,N,N′,N′-tetraisopropyl-phosphora-
midite (55 µL, 52 mg, 0.174 mmol) were added. The reaction
mixture was stirred for 3 h at room temperature under an argon
atmosphere. The solvent was removed in vacuo, and the crude
mixture of the mono- and bis-phosphatidylated products was
dissolved in CH3OH (15 mL) and stirred for 30 min at room
temperature. The solvent was removed in vacuo, and the residue
was purified by flash column chromatography on silica gel, eluting
with a gradient of CH2Cl2/CH3OH/pyridine (97:1:2 to 94:4:2, v/v/
v) to afford phosphoramidite 6 (60 mg, 55% yield). 1H NMR (400
MHz, DMSO-d6) δ 7.96 and 7.95 (s, 1H, diastereomeric H-2), 7.32
(m, 2H, aromatic), 7.20 (m, 7H, aromatic), 7.16 and 7.14 (s, 1H,
diastereomeric H-6), 6.78 (m, 4H, aromatic), 6.26 and 6.25 (m,
1H, diastereomeric H-1′), 4.11 (s, 2H, H-10), 4.07 and 4.02 (s, 1H,
diastereomeric H-4′), 3.73 and 3.62 (m, 2H, POCH2), 3.70 and 3.69
(s, 6H, 2OCH3), 3.52 (m, 2H, CH(CH3)2), 3.21 (m, 2H, H-5′, H-5′′),
2.84 (m, 1H, H-2′), 2.75 (t, 1H, J ) 5.9 Hz, CH2CN), 2.65 (t, 1H,
J ) 5.9 Hz, CH2CN), 2.55 (t, 2H, J ) 7.3 Hz, H-12), 2.42 (m, 1H,
H-2′′), 1.50 (m, 2H, H-13), 1.25 (m, 4H, H-14, H-15), 1.12 and
1.02 (m, 12H, 2CH(CH3)2), 0.85 (t, 3H, J ) 6.8 Hz, H-16). 13C
NMR (150 MHz, DMSO-d6) δ 206.0, 158.5, 154.1, 150.3, 150.0,
147.2, 145.1, 137.0, 135.9, 135.8, 135.7, 130.1, 128.1, 128.0, 127.1,
124.3, 119.4, 119.2, 118.2, 116.4, 113.5, 86.1, 85.2, 84.9, 83.1,
83.0, 73.9, 73.4, 64.0, 58.9, 58.8, 58.7, 58.6, 55.4, 43.1, 41.6, 31.2,
24.8, 24.8, 24.7, 24.7, 24.6, 24.6, 23.1, 22.4, 20.3, 20.2, 20.1, 14.3.
31P NMR (400 MHz, DMSO-d6) δ 149.15, 148.5.
Capillary Gel Electrophoresis (CGE). Electrophoretic analyses
were carried out using a Beckman P/ACE MDQ instrument system
(using 32 Karat software, version 5.0) monitored at 260 nm on a
31.2 cm × 100 µm eCAP capillary with samples applied at 10 kV
and run at 9 kV. The capillary was packed with their 100-R gel
(for ss-DNA) using the Tris-borate buffer system containing 7 M
urea. The purity of the oligonucleotides was judged to be >99%
(see Figures S4-S6 of Supporting Information).
Oligonucleotide Labeling and Annealing. The labeling and
annealing of the oligonucleotides were performed as previously
described (40).
Single-Nucleotide Incorporation Assays. These assays were
performed as previously described with the following modifications
(40). The reactions with Dpo4 (35) were initiated by the addition
of dNTP with final concentrations of 30, 60, and 120 µM. The
final concentration of Dpo4 was 100 nM, and the reactions were
run at 37 °C for 30 min. The reactions with the primer and
unmodified template were run under the same reaction conditions
but with half the enzyme concentration. Reactions were quenched
with 70 µL of 20 mM EDTA in 95% formamide (v/v) containing
xylene cyanol and bromophenol blue dyes, and heated at 95 °C for
10 min. Aliquots (6 µL) were separated by electrophoresis on a
denaturing gel.
Steady-State Kinetics. These assays were performed according
to a published procedure with the following modifications (40).
All reactions (10 µL final volume, 100 nM DNA, 100 nM Dpo4)
were run at nine dNTP concentrations. The reactions were quenched
and analyzed by gel electrophoresis as described above.
Full-Length Polymerase Extension Assays. These assays were
performed as previously described with the following modifications
(40). The reactions were run with 100 nM DNA and 100 nM Dpo4
at 37 °C for 30 min. The final dNTP concentrations were 30, 60,
and 120 µM each. The reactions with the primer and unmodified
template were run using the same reaction conditions but with half
the enzyme concentration. The reactions were quenched and
analyzed by gel electrophoresis as described above.
FAB HRMS+ m/z calcd for C49H61N7O8P [M + H]+, 906.4319;
found, 906.4309.
Oligonucleotide Synthesis. Modified oligonucleotides were
synthesized on a Perseptive Biosystems Model 8909 DNA synthe-
sizer using Expedite reagents (Glen Research, Sterling, VA) with
the standard synthetic protocol on a 1-µmol scale. Oligonucleotides
containing the 7-(2-oxoheptyl)-ε-dGuo were prepared using a
standard DNA synthesizer cycle with p-(t-butylphenoxy)acetyl-
protected phosphoramidites. The modified oligodeoxynucleotides
were cleaved from the solid support, and the exocyclic amino groups
were deprotected in a single step using 0.1 M aq NaOH; the
cleavage reaction was neutralized and the oligonucleotides purified
by HPLC (gradient A). All unmodified oligonucleotides were
purchased from Midland Certified Reagents (Midland, TX).
5′-TCA TXG AAT CCT TAC GAG CCC CC-3′ (7). Solid-
phase synthesis on 1-µmol scale afforded 31 A260 units (135 nmol).
MALDI-TOF-MS (HPA) m/z calcd for [M - H]- 7080.7; found,
7080.5.
LC-ESI-MS-MS Sequencing of the Extension Reactions.
Extension of templates 7 and 8 and identification of the products
by LC-ESI-MS-MS were accomplished as previously described
(36). Mass spectrometric analyses were performed in the Vanderbilt
University facility on a Waters Acquity UPLC system (Waters,
Milford, MA) connected to a Finnigan LTQ mass spectrometer
(ThermoElectron) equipped with an Ion Max API source and a
standard electrospray probe using an Acquity UPLC BEH C18
column (1 µm, 1.0 mm × 100 mm). These analyses were performed
as previously described (36). The Mongo Oligo Mass Calculator
(v. 2.6) from the University of Utah (http://library.med.utah.edu/
masspec/) was used to calculate the theoretical CID spectra of the
candidate oligonucleotide sequences. The proposed oligonucleotide
sequences were then purchased from Midland Certified Reagents
(Midland, TX) and subjected to the same LC-ESI-MS-MS analysis
in order to compare the CID spectra.
5′-TCA CXG AAT CCT TAC GAG CCC CC-3′ (8). Solid-
phase synthesis on 1-µmol scale afforded 36 A260 units (158 nmol).
MALDI-TOF-MS (HPA) m/z calcd for [M - H]- 7065.7; found,
7065.6.

Bypass of 7-(2-Oxoheptyl)-ε-dGuo by Dpo4
Chem. Res. Toxicol., Vol. 23, No. 8, 2010 1333
Construction of the Calibration Curves. Standard calibration
curves were constructed using 5-7 different concentrations (from
0.02 to 0.40 nmol) of the corresponding oligonucleotide (analyte)
and a constant amount of the 5′-pCTTCACGAGCCCCC-3′ standard
(0.10 nmol) (36). The amount of the standard corresponded to
∼50% yield for the extension reaction. The yield of formation of
the corresponding analyte was calculated on the basis of the ratio
(nmol) of the extension product and the biotinylated primer (nmol)
used for the full-length extension reaction. The correlation coef-
ficients (r2) were typically >0.98. The calibration curves were
constructed in full scan mode using the sum of the extracted [M -
2H]-2 and [M - 3H]-3 ions for the analyte oligonucleotides and
the sum of the [M - 3H]-3 and [M - 4H]-4 ions for the standard.
Each peak consisted of at least 15 scans. Calibration curves are
shown in Figures S23 and S24 of the Supporting Information.
To determine the accuracy of the calibration curves, a mixture
of the 5′-pCTTACGAGCCCCC-3′ standard (0.10 nmol) was added
to known amounts of the 5′-pTCCGTGA-3′ (0.06 nmol), 5′-
pTCAGTGA-3′ (0.18 nmol), 5′-pTCTGTGA-3′ (0.40 nmol), and
5′-pTCGTGA-3′ (0.08 nmol) analyte oligonucleotides, and the
mixture volume was brought to 20 µL; 10 µL of this solution was
analyzed. The amounts of the analyte oligonucleotides were
calculated on the basis of the ratio of the area of analyte to standard
(5′-pCTTACGAGCCCCC-3′) and the corresponding calibration
curve. The calculated amount of the analyte oligonucleotides was
generally within 10% of the actual amount.
Scheme 1
The synthesis of 6 required 5 steps from dGuo and ONE and
proceeded in ∼18% overall yield.
Phosphoramidite 6 was used for the syntheses of three
oligonucleotides (7-9); standard solid-phase protocols were
used and the oligonucleotides characterized by MALDI-TOF-
MS. The modified oligonucleotides were purified by HPLC, and
their purity was >99% based on capillary gel eletrophoretic
analysis (see Figures S4 and S5 of the Supporting Information).
The 23-mer oligonucleotides (7 and 8, see Figure 2 for
sequences) were synthesized for in vitro replication studies with
Dpo4, and the 12-mer oligonucleotide, 5′-CGCATXGAATCC-
3′ (9), was synthesized for structural studies.
UV Melting Studies. Experiments were conducted on a Cary
100 Bio spectrophotometer (Varian Associates, Palo Alto, CA) with
∼0.3 A260 units of duplex sample in 1 mL of 10 mM NaH2PO4,
100 mM NaCl, and 5 µM Na2EDTA (pH 7.0) in a 1.0 cm cuvette.
Experiments were also conducted at pH 5.2 and pH 8.6. Samples
were heated at a rate of 2 °C/min, and data were collected at a rate
of 1 point/min.
NMR Experiments. The modified duplex, 5′-d(CGCATX-
GAATCC)-3′ (9) and 5′-d(GGATTCCATGCG)-3′, where X )
7-(2-oxoheptyl)-ε-dGuo, was prepared as previously described for
the 1,N2-ε-dGuo containing duplex (37, 38). Briefly, samples for
NMR measurements were dissolved to a duplex concentration of
0.3 mM in 500 µL of 10 mM NaH2PO4, 100 mM NaCl, and 5 µM
Na2EDTA (pH 7.0). Samples for the observation of nonexchange-
able protons were exchanged with D2O and suspended in 500 µL
of 99.99% D2O. Samples for the observation of exchangeable
protons were dissolved to a concentration of 0.3 mM in 500 µL of
10 mM NaH2PO4, 100 mM NaCl, and 5 µM Na2EDTA containing
9:1 H2O/D2O. The desired pH values of the NMR samples were
obtained by titration with concentrated DCl or NaOD. The
temperature was 20 °C for the observation of nonexchangeable
protons and 5 °C for the observation of exchangeable protons.
Chemical shifts of the proton resonances were referenced to the
water resonance at 4.82 ppm at 20 °C and 4.96 ppm at 5 °C. NMR
experiments and analysis were carried out as described before (37).
Insertion Opposite 7-(2-Oxoheptyl)-ε-dGuo by Dpo4 in
the Presence of a Single dNTP. The 7-(2-oxoheptyl)-ε-dGuo
Results
Phosphoramidite and Oligonucleotide Synthesis. We have
reported an improved synthesis of the phosphoramidite reagent
of 1,N2-ε-dGuo (41). A key to this work was the recognition
that the N5-position was reactive (42). 7-(2-Oxoheptyl)-ε-dGuo
(1) was prepared by the reaction of ONE with dGuo by a slightly
modified procedure in 55% yield (7). Modified nucleotides
containing a ketone have been incorporated into oligonucleotides
previously using the phosphoramidite approach indicating that
this functional group is compatible with solid-phase synthesis
(32, 43). Reaction of 1 with excess dimethoxytrityl chloride
(DMTr-Cl) afforded 5′,N5-bis-dimethoxytritylated product 4
(Scheme 1). The N5-DMTr group was selectively removed by
briefly heating 4 in CH3OH at reflux. Phosphitylation of 5 gave
the 3′,N5-bis-phosphoramidite reagent, which was not character-
ized. The crude product was stirred in methanol at room
temperature to afford the desired phosphoramidite reagent 6.
Figure 2. Full-length extension of the 7-(2-oxoheptyl)-ε-dGuo lesion
in sequences 7 (top) and 8 (bottom). Reaction times were 30 min for
the modified oligonucleotides (X ) 7-(2-oxoheptyl)-ε-dGuo) and 5 min
for the unmodified control (X ) dGuo).

1334 Chem. Res. Toxicol., Vol. 23, No. 8, 2010
ChristoV et al.
Table 1. Steady-State Kinetic Parameters for Single-Nucleotide Incorporation Opposite the 7-(2-Oxoheptyl)-ε-dGuo Adduct in
Oligonucleotides 7 and 8 by Dpo4
template
dNTP
Km (µM)
kcat (min-1
)
kcat/Km (µM-1·min-1
)
f a
5′---T-[7-(2-oxoheptyl)-
ε-dGuo]-G---3′ (7)
C
T
G
A
C
T
G
A
C
T
G
A
C
T
85 ( 12
62 ( 10
120 ( 23
7.6 ( 1.2
4.7 ( 0.4
22 ( 4
0.010 ( 0.004
0.014 ( 0.09
0.011 ( 0.001
0.029 ( 0.008
6.4 ( 0.13
0.069 ( 0.001
0.042 ( 0.003
0.12 ( 0.01
0.043 ( 0.002
0.025 ( 0.001
0.050 ( 0.003
0.090 ( 0.006
0.053 ( 0.02
0.099 ( 0.003
0.053 ( 0.001
0.090 ( 0.006
1.2 × 10-4
2.3 × 10-4
9.2 × 10-5
3.8 × 10-3
1.4
1.0
1.9
0.8
32
5′---T-G-G---3′
1.0
3.1 × 10-3
5.3 × 10-4
3.6 × 10-3
3.2 × 10-4
1.4 × 10-3
1.4 × 10-3
5.6 × 10-3
19
2.2 × 10-3
3.7 × 10-4
2.6 × 10-3
1.0
80 ( 17
33 ( 8
5′---C-[7-(2-oxoheptyl)-
ε-dGuo]-G---3′ (8)
135 ( 25
18 ( 3
35 ( 8
16 ( 3
0.028 ( 0.003
61 ( 9
78 ( 6
230 ( 42
4.4
4.4
18
1.0
5′---C-G-G---3′
1.6 × 10-3
6.8 × 10-4
3.9 × 10-4
8.4 × 10-5
3.6 × 10-5
2.1 × 10-5
G
A
a f ) misincorporation frequency ) (kcat/Km)incorrect dNTP/(kcat/Km)correct dNTP (dCTP)
.
adduct was incorporated at position 5 of the 23-mer oligonucle-
otides (7 and 8); these substrates were then annealed to the
complementary 5′-32P-labeled 18-mer (-1) primer strand for
single nucleotide incorporation studies. Oligonucleotides 7 and
8 differ in the identity of the base on the 5′-side of the lesion
(Thy vs Cyt). In general, the efficiency for dNTP insertion
opposite the 7-(2-oxoheptyl)-ε-dGuo lesions was low, and the
pattern of dNTP insertion was similar to that of 1,N2-ε-dGuo
(see Figure S9 of the Supporting Information). Insertion of dATP
appears to be favored for both etheno adducts in both sequences,
and this was confirmed by steady-state kinetic analysis (Table
1). The successive insertion of three dAdo nucleotides was
observed for the 5′-TXG-3′ (7) template, which was also
reported for 1,N2-ε-dGuo in the same sequence context (35).
Only dCTP was inserted opposite dGuo of the unmodified
control templates. Insertion of dCTP opposite the lesion was
g104 less efficient (kcat/Km) than that for the unmodified template
in both sequences. Incorporation of dATP opposite 7-(2-
oxoheptyl)-ε-dGuo was most efficient with kcat/Km values 18-
and 32-fold greater than the insertion of dCTP for the 5′-TXG-
3′ (7) and 5′-CXG-3′ (8) templates, respectively. Insertion of
dTTP and dGTP was 16- and 41-fold less efficient than the
insertion of dATP for the 7-(2-oxoheptyl)-ε-dGuo lesion in the
5′-TXG-3′ (7) template and 17- and 4-fold less efficient in
the 5′-CXG-3′ (8) template; the catalytic efficiencies were
similar to the misinsertion opposite an unmodified template (X
) dGuo).
Figure 3. Full scan product ion mass spectra for the bypass and
extension of the 7-(2-oxoheptyl)-ε-dGuo lesion in the 5′-TXG-3′ (7,
top) and 5′-CXG-3′ (8, bottom) templates. Sequence assignments are
listed in Table 2. The CID mass spectra of each extension product can
be found in Supporting Information (Figures S10-S22).
beads by treatment with uridine DNA glycosylase followed by
piperidine (see Figure S8 of the Supporting Information). The
resulting oligonucleotides, which were 7-mers for full-length
extension products, were sequenced by LC-ESI-MS-MS. A 13-
mer oligonucleotide standard was added just prior to MS
analysis in order to better estimate the chemical yield of the
extension products. We assumed that the remainder of the mass
balance was unextended primer or truncated extension products,
which were not observed.
Translesion Synthesis Across 7-(2-Oxoheptyl)-ε-dGuo
by Dpo4. The 5′-TXG-3′ (7) and 5′-CXG-3′ (8) templates were
annealed to a complementary 16-mer (-3) primer strand, and
the ability of Dpo4 to extend past the 7-(2-oxoheptyl)-ε-dGuo
lesion in the presence of all four dNTPs was examined (Figure
2). Full-length products were observed in both sequences after
a 30 min reaction time indicating that Dpo4 could efficiently
bypass the lesion.
Characterization of Extension Products by MS. We
previously characterized extension products from in vitro
translesion synthesis by LC-ESI-MS-MS (35), and an improve-
ment of this method was recently reported (36). Briefly, a 5′-
biotinylated, -1 primer with a dUrd at the -3 position was
hybridized to the templates (see Figure S7 of the Supporting
Information for primer sequence). Immobilization of the ex-
tended primers on strepavidin-coated beads allowed for the
partial purification and enrichment of the extension products,
which translated into greater sensitivity during MS analysis. The
immobilized extension products were then cleaved from the
We used this method to sequence the extension products from
bypass of the 7-(2-oxoheptyl)-ε-dGuo lesion, and the 1,N2-ε-
dGuo adduct was reexamined in the same sequences for
comparison. The full scan product ion mass spectra from
translesion synthesis of the modified 5′-TXG-3′ (7) and 5′-CXG-
3′ (8) templates are shown in Figure 3, and the sequence
assignments of the extension products are summarized in
Table 2.
The same seven extension products were identified for the
bypass of both etheno lesions in the 5′-TXG-3′ (7) template,

Bypass of 7-(2-Oxoheptyl)-ε-dGuo by Dpo4
Chem. Res. Toxicol., Vol. 23, No. 8, 2010 1335
Table 2. Summary of the Bypass and Extension of the 7-(2-Oxoheptyl)-ε-dGuo and ε-dGuo Lesions in 5′-TXG-3′ (7) and
5′-CXG-3′ (8) Templates by Dpo4 as Determined by LC-ESI-MS-MS Sequencing
although their distribution was slightly different. Misinsertion
of Ade (5′-pTCAATGA-3′, ∼35%) and a one-base deletion (5′-
pTC-ATGA-3′, ∼20%) were the major extension products when
the 7-(2-oxoheptyl)-ε-dGuo lesion was bypassed. A minor
product (5′-pTC-ATG-3′, ∼4%) was identified as also arising
from a one-base deletion, but incomplete extension (vide infra).
Other minor products (<5% each) were derived from error-free
bypass (5′-pTCCATGA-3′) and the misinsertion of Gua (5′-
pTCGATGA-3′).
consistent with the w4 fragment ions of the 5′-pTCATTGA-3′
product. The CID spectrum of an authentic standard of the 5′-
pTCTATGA-3′ oligonucleotide showed the a4-b fragment ion
at m/z 1074.1 but not the w4 fragment ion at m/z 1276.2, similar
to the spectrum from the extension reaction. Additionally, the
fragment ions at m/z 1083.1 and 1267.2 were not observed. The
w4 fragment ions at m/z 1267.1 and 633.1 ([M - 2H]-2) were
observed in the CID spectrum of an authentic standard of the
5′-pTCATTGA-3′ sequence, while the a4-b fragment ion was
not observed (m/z 1083.1); as expected, no fragment ions were
observed at m/z 1074.1 and 1276.2.
Two major products from translesion synthesis of the 1,N2-
ε-dGuo lesion derived from the misinsertion of Ade (16%) and
a one-base deletion (31%) were identified. The minor products
(<5%) were identified as the error-free bypass product, misin-
sertion of Gua, misinsertion of Thy, and a tandem misinsertion
(5′-pTCATTGA-3′). The latter two were observed as a mixture,
as discussed above.
We previously reported that the 1,N2-ε-dGuo lesion in the
5′-TXG-3′ sequence resulted in four products including the
misinsertion of Ade (5′-pTCAATGA-3′), a tandem misinsertion
(5′-pTCATTGA-3′), a one-base deletion (5′-pTC-ATGA-3′), and
a two-base deletion (5′-pTC- -TGA-3′) (35). The products
arising from misinsertion of Ade and a one-base deletion were
observed in the current analysis. As discussed above, the tandem
misinsertion product was a mixture with the isomeric misin-
sertion of Thy (5′-pTCTATGA-3′). Upon closer examination
of the CID spectrum, the sequence assignment for the two-base
deletion product was revised to the isomeric 5′-pTC-ATG--3′
(35), which arises from a one-base deletion opposite the etheno-
lesion and truncation of the extension reaction one-base short
of full-length. The proposed two-base deletion product is
expected to have w1 and w2 fragment ions at m/z 330.1 and
659.1, respectively, and an a4-B fragment ion at m/z 1074.1 in
the CID mass spectrum, whereas these same fragment ions for
the 5′-pTC-ATG--3′ product are predicted for m/z 346.1, 650.1,
and 1083.1, respectively (Figure 5). The observed CID spectrum
of the extension product (m/z 777.3, [M - 2H]-2) showed a
prominent fragment ion at m/z 650.0 and a less abundant ion at
m/z 1083.2, consistent with the one-base deletion product and
incomplete extension (5′-pTC-ATG--3′); fragment ions unique
Analysis of the CID spectrum of the m/z 1086.2 [M - 2H]-2
ion indicated that two products of the same nucleotide composi-
tion were present, one resulting from the misinsertion of Thy
(5′-pTCTATGA-3′) and a tandem misinsertion (5′-pTCATTGA-
3′) involving incorporation of Ade opposite the lesion followed
by misinsertion of the Thy opposite the template Thy that is 3′
to the lesion. The CID spectra from the two sequences are
predicted to be very similar (Figure 4). The only differences
are the a4-b and w4 [M - H]- fragment ions, which are predicted
to give m/z 1074.1 and 1276.2 for the 5′-pTCTATGA-3′
oligonucleotide and m/z 1083.1 and 1267.2 for the 5′-pTCAT-
TGA-3′ sequence (Figure 4). A prominent fragment ion at m/z
1074.6 was observed to be consistent with the a4-b fragment
ion of the 5′-pTCTATGA-3′ product (see Figure S14 of the
Supporting Information); however, the w4 fragment ion at m/z
1276.2 was not observed. Less abundant ions were observed at
m/z 1268.0 [M - H]- and m/z 634.1 [M - 2H]-2 and are
Figure 4. Predicted CID fragment [M - H]- ions for the 5′-
pTCTATGA-3′ and 5′-pTCATTGA-3′ extension products (m/z 1086.2,
[M - 2H]-2). The experimental CID spectra are found in Figure S14
of Supporting Information.

1336 Chem. Res. Toxicol., Vol. 23, No. 8, 2010
ChristoV et al.
dGuo and 1,N2-ε-dGuo lesions lowered the Tm of the duplex
by 13-20 °C compared to that of the unmodified duplex. The
7-(2-oxoheptyl)-ε-dGuo was more destabilizing than the 1,N2-
ε-dGuo lesions by 1-7 °C depending on the pH. The Tm of the
unmodified duplex (52-53 °C) and 7-(2-oxoheptyl)-ε-dGuo
containing duplex (33-34 °C) was not sensitive to pH. The Tm
of the duplex containing the 1,N2-ε-dGuo lesion was 4-5 °C
less stable at basic pH (35 °C) than under acidic (40 °C) or
neutral (39 °C) conditions. The 7-(2-oxoheptyl)-ε-dGuo lesion
decreased the thermal stability of the duplex at acidic and neutral
pH compared to that of 1,N2-ε-dGuo lesion (∆Tm 7 and 5 °C,
respectively). Interestingly, the Tm of the 7-(2-oxoheptyl)-ε-dGuo
containing duplex remained constant when the base opposite
the lesion was Cyt, Ade, or a one-base deletion. This is in
contrast to the 1,N2-ε-dGuo containing duplex, which was
slightly more stable when Ade or a one-base deletion was
opposite the lesion.
Figure 5. Predicted CID fragment [M - H]- ions for the 5′-pTC-ATG-
-3′ and 5′-pTC- -TGA-3′ extension products (m/z 777.3, [M - 2H]-2).
Table 3. Melting Temperatures (Tm, Expressed in °C) of
Duplex DNA Containing the 7-(2-Oxoheptyl)-ε-dGuo and
1,N2-ε-dGuo lesions, and the Unmodified dGuo in the
5′-CGCATXGAATCC-3′ (9)·5′-GGATTCYATGCG-3′ DNA
Duplexes at Acidic, Neutral, and Basic pH Values (10 mM
NaH2PO4, 100 mM NaCl, and 5 µM Na2EDTA)
NMR Analysis of 7-(2-Oxoheptyl)-ε-dGuo Opposite dCyd
in Duplex DNA. At 5 °C, the far downfield region of the proton
NMR spectra of the 7-(2-oxoheptyl)-ε-dGuo containing duplex
obtained either at pH 7 or pH 8.6 appeared similar (Figure 6).
The 7-(2-oxoheptyl)-ε-dGuo base lacks an imino proton due to
its alkylation at the N8 position (this is N1 in guanine
numbering). The imino proton resonances of the unmodified
Thy and Gua bases were assigned from NOESY spectra (vide
infra). The imino resonance of the 5′-neighbor base pair T5:A20
was not observed, and that of the 3′-neighbor base pair G7:C18
was broad, indicating increased exchange with water (Figure
S25 in the Supporting Information). At pH 7, all of the imino
proton resonances broadened between 40-45 °C, whereas at
pH 8.6, all of the imino proton resonances broadened between
35-40 °C. The lack of dependency of the Tm of the 7-(2-
oxoheptyl)-ε-dGuo containing duplex on pH, which was also
observed for the unmodified duplex, was attributed to base-
catalyzed exchange in the presence of buffer (Figure S26 in
the Supporting Information). Figure 6 shows contour plots of
expanded regions of the NOESY spectrum showing NOE
connectivities between the imino protons (bottom) and between
the imino and amino or Ade H2 (top) protons. At pH 7 and pH
8.6, the expected pattern of NOE cross-peaks was observed for
the G2·C23, C3·G22, and A4·T21 base pairs, located in the 5′
direction from the 7-(2-oxoheptyl)-ε-dGuo adduct, except
between A4·T21 and T5·A20 (Figure 6A-C). Likewise, NOE
connectivities were observed between base pairs G7·C18, A8·T17,
A9·T16, T10·A15, and C11·G14, located in the 3′-direction from the
7-(2-oxoheptyl)-ε-dGuo adduct. The N5H amino proton of 7-(2-
oxoheptyl)-ε-dGuo was not observed, presumably because of
exchange. Other aspects of the NOESY spectra of the 7-(2-
oxoheptyl)-ε-dGuo containing duplex acquired at pH 7.0 and
pH 8.6 were also similar (Figure 6B and C). Sequential
nucleotide assignments were made using standard protocols
(44, 45). The sequential NOE connectivity in the modified strand
was observed from C1 through T5 H1′. No NOEs between 7-(2-
oxoheptyl)-ε-dGuo (X6) and its 3′ neighbor G7 were observed,
indicative of disorder. The entire sequential NOE connectivity
for the complementary strand was observed from G13fG24.
However, the C18 H1′fC19 H6 (observed at pH 8.6), C19
H6fC19 H1′, and C19 H1′fA20 H8 NOEs were weak. The C19
H6fC19 H5 cross-peak was broad. The heptyl chain proton
resonances were assigned at neutral and basic pH values. No
NOEs arising between the heptyl protons and DNA were
observed.
lesion (X)
Y
pH 5.2
pH 7.0
pH 8.6
7-(2-oxoheptyl)-ε-Gua
C
A
33 ( 1
34 ( 1
34 ( 1
34 ( 1
39 ( 1
43 ( 1
45 ( 1
53 ( 1
34 ( 1
1,N2-ε-Guaa
C
A
40 ( 1
35 ( 1
Guaa
C
53 ( 1
52 ( 1
a The melting temperatures were previously reported (37, 38).
to the two-base deletion product were not observed (see Figure
S16 in the Supporting Information).
Five extension products were observed for the Dpo4 bypass
of the 7-(2-oxoheptyl)-ε-dGuo lesion in the 5′-CXG-3′ template
(8). The major products were derived from misinsertion of Ade
(5′-pTCAGTGA-3′, 26%), a one-base deletion (5′-pTC-GTGA-
3′, 20%), and error-free bypass (5′-pTCCGTGA-3′, 14%). Minor
products (<5%) resulted from the misinsertion of Gua (5′-
pTCGGTGA-3′) and Thy (5′-pTCTGTGA-3′). Four extension
products were characterized from the bypass of the 1,N2-ε-dGuo
lesion in this sequence. The major products were error-free
bypass (∼33%) and a one-base deletion (24%). The two minor
products resulted from the misinsertion of Ade (∼8%) and Gua
(∼3%). The product derived from the misinsertion of Thy was
below the limit of MS detection and not observed.
Quantitation of the extension products by MS was based on
calibration curves developed for the response of a single
extension product analyte versus a 13-mer standard (5′-
pCTTCACGAGCCCCC-3′). Chromatographic separation of the
extension products was not achieved. Quantitation errors were
therefore introduced since the ionization efficiency of the
coeluting analytes is expected to be different from that of a
single component. A sample containing known quantities of the
extension products from the 5′-CXG-3′ template and the 13-
mer standard was analyzed by LC-ESI-MS-MS to estimate the
quantitation error. The quantitation was generally within 10%
error (36).
Characterization of the 7-(2-Oxoheptyl)-ε-dGuo Adduct
in Duplex DNA Opposite dCyd. The UV thermal melting
temperatures (Tm) of the 5′-CGCATXGAATCC-3′ (9)·5′-
GGATTCCATGCG-3′ duplexes where X is 7-(2-oxoheptyl)-
ε-dGuo, 1,N2-ε-dGuo, and dGuo at pH 5.2, 7.0, and 8.6 are listed
in Table 3. The local sequence of the modified base is related
to oligonucleotide 7. The presence of the 7-(2-oxoheptyl)-ε-
The proton NMR spectrum of the 7-(2-oxoheptyl)-ε-dGuo
containing duplex differed at 5 °C and pH 5.2. The G7 N1H

Bypass of 7-(2-Oxoheptyl)-ε-dGuo by Dpo4
Chem. Res. Toxicol., Vol. 23, No. 8, 2010 1337
1
Figure 6. H NOESY spectra showing the sequential NOE connectivity between the imino protons (bottom) and NOEs between imino and the
amino and base protons (top) of the 7-(2-oxoheptyl)-ε-dGuo·dCyt duplex at (A) pH 5.2, (B) pH 7.0, and (C) pH 8.6. Labeled peaks are a, T17
N3HfA9 H2; b, T16 N3HfA8 H2; c, T10 N3HfA15 H2; d, T21 N3HfA4 H2; e,e′, G2 N1HfC23 N4H, h/n; f,f′, G14 N1HfC11 N4H, h/n; g,g′, G22
N1HfC3 N4H, h/n; h,h′, G7 N1HfC18 N4H, h/n. The symbols h and n stand for hydrogen bonded and non-hydrogen bonded amino protons of
cytosine.
proton resonance remained broadened (Figure S25 in the
Supporting Information). Additional line broadening involving
the imino proton resonances at base pairs A8:T17, and A9:T16
was attributed to increased exchange with water at these two
base pairs at the lower pH value (Figure S25 in the Supporting
Information). The NOE connectivity between the G7·C18 and
A8·T17 base pairs was missing. (Figure 6, labeled as G7). Very
weak NOE cross-peaks were observed between the G7 N1H and
the amino protons from the complementary Cyt (C18). The
resonances for the C19 amino protons were not observed,
suggesting rapid exchange with water. The anticipated pattern
of sequential base aromatic proton to deoxyribose H1′ NOEs
was identified from nucleotide C1 through nucleotide A4 (Figure
7A). The A4 H1′fT5 H6 and T5 H6fT5 H1′ NOEs were weak
and not observed at the threshold plotted in Figure 7A. The
anticipated NOEs involving the lesion, T5 H1′fX6 H2 (H2 is
the imidazole proton of 7-(2-oxoheptyl)-ε-dG; see Figure 1 for
atom numbering), X6 H2fX6 H1′, and X6 H1′fG7 H8, were
missing, and the NOE connectivity was interrupted. The G7
H8fG7 H1′ NOE was weak. Proceeding in the 3′-direction from
the G7 H8fG7 H1′ NOE, the NOESY connectivities continued
uninterrupted to the 3′-terminous of the modified strand. The
NOE connectivities for the complementary strand were continu-
ous from G13 through C19, which is the complementary nucle-
otide to the lesion X6 (Figure 7A). However, the C19 resonances
were broad. The C19 H6fC19 H1′ and C19 H1′fA20 H8 NOEs
were missing. Proceeding in the 3′-direction from the A20
H8fA20 H1′ NOE, the NOESY connectivities continued
uninterrupted to the 3′-terminous of the modified strand. The
pattern of the NOEs between the base aromatic protons and
the deoxyribose H2′ and H2′′ protons was similar to that
observed for the NOEs between the aromatic protons and
deoxyribose H1′ protons, in both the modified and complemen-
tary strands (data not shown). Because of the line broadening,
it was not possible to assign the heptyl chain proton resonances
at acidic pH.
Discussion
Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a
member of the Din B family of DNA polymerases. Din B
polymerases are found in all kingdoms of life and include
Escherichia coli pol IV and human pol κ. The specialized
function of Din B polymerases is to replicate through damaged
DNA (46, 47). Dpo4 was noted to have lesion bypass properties
similar to those of human pol η, which is part of the Rad30
family of lesion bypass polymerases (48). Recombinant Dpo4
can be expressed at high levels in E. coli and has been
crystallized with DNA substrates containing the 1,N2-ε-dGuo
(35), 8-oxo-dGuo (49-52), cis-syn thymine dimer (53), O6-
alkyl-dGuo (54, 55), the tetrahydrofuran analogue of an abasic
site (56), a malondialdehyde adduct of dGuo (M1dGuo) (57),
and N2-dGuo and N6-dAdo adducts of benzo[a]pyrene diol
epoxide (BPDE) (58, 59).
We reported the in vitro replication of 1,N2-ε-dGuo by Dpo4
(35). The misincorporation profile was dependent on the 5′-
neighboring base relative to the lesion. Replication of 1,N2-ε-
dGuo by Dpo4 resulted in largely a one-base deletion when a
dCyd was 5′ to the modified base (template 8); a minor product
derived from misincorporation of dATP was also observed. In
the current work, we identified two additional products from
the bypass of the 1,N2-ε-dGuo adduct in the 5′-CXG-3′ template
(8) by Dpo4. These were derived from the misinsertion of Gua
(5′-pTCGGTGA-3′, 3%) and error-free bypass (5′-pTCCGTGA-
3′, 33%). Replication of the 7-(2-oxoheptyl)-ε-dGuo lesion in
the same sequence resulted in five extension products. In
addition to the same four products from the bypass of the 1,N2-
ε-dGuo lesion, a minor product identified as the misinsertion
of Thy resulted from the replication of the 7-(2-oxoheptyl)-ε-

1338 Chem. Res. Toxicol., Vol. 23, No. 8, 2010
ChristoV et al.
Figure 7. 1H NOESY spectra showing the NOE connectivity from anomeric H1′ to 3′-neighbor aromatic protons of the 7-(2-oxoheptyl)-ε-dGuo·dC
duplex at (A) pH 5.2, (B) 7.0, and (C) 8.6 (20 °C). Asterisks indicate the cytosine H5-H6 cross-peaks. The dotted line indicates that the NOE
cross-peak between C18 and C19 was observed at a lower threshold level.
Scheme 2
dGuo adduct (5′-pTCTGTGA-3′, 4%). The overall yield for the
bypass of the two etheno lesions was very similar in the 5′-
CXG-3′ template sequence, but the distribution of products
differed. Replication of the 7-(2-oxoheptyl)-ε-dGuo lesion
resulted in a lower frequency of the error-free product than did
1,N2-ε-dGuo (14% vs 33%), and a significantly larger proportion
of the product distribution resulted from misinsertion of Ade
(26% vs 8%). Replication of both etheno lesions by Dpo4
resulted in a one-base deletion product.
Replication of the 1,N2-ε-dGuo and 7-(2-oxoheptyl)-ε-dGuo
lesions in the 5′-TXG-3′ template sequence (7) resulted in the
same seven extension products, and the total yield of extension
products was similar for the two adducts. Replication of the
etheno adducts was considerably more error-prone in the 5′-
TXG-3′ template than the 5′-CXG-3′ sequence with <5% of
the extension products resulting from error-free bypass. The
major extension products from the bypass of both etheno lesions
in the 5′-TXG-3′ template (7) were derived from a one-base
deletion (5′-pTC-ATGA-3′ and 5′-pTC-ATG--3′) and misin-
sertion of Ade (5′-pTCAATGA-3′). In the case of the 1,N2-ε-
dGuo lesion, the one-base deletion product predominated by
an ∼2:1 ratio, whereas bypass of the 7-(2-oxoheptyl)-ε-dGuo
lesion resulted in a higher proportion of misinsertion of Ade
(∼1:1.5); this same trend was also observed for the 5′-CXG-3′
sequence. Two additional minor products were indentified
(<5%), which arise from the misinsertion of Gua (5′-TC-
GATGA-3′) and error-free bypass (5′-pTCCATGA-3′).
noninstructive insertion of dATP opposite 7-(2-oxoheptyl)-ε-
Gua (Scheme 2). Extension of the misaligned or aligned
primer-template complex leads to the one-base deletion or
misinsertion of Ade products, respectively. It is possible that
the aligned and misaligned primer-template can interconvert.
The catalytic efficiency for dATP insertion opposite the 7-(2-
oxoheptyl)-ε-dGuo lesion in the 5′-CXG-3′ template was slightly
higher than that of the 5′-TXG-3′ sequence. The modest
sensitivity to the 5′-neighboring base (Thy vs Cyt) suggests a
noninstructive insertion of dATP opposite the 7-(2-oxoheptyl)-
ε-dGuo lesion since it is unlikely that dATP will pair with 5′-
Cyt of the 5′-CXG-3′ sequence. This indicates that the one-
base deletion product may arise from a misincorporation-
misalignment mechanism for the 7-(2-oxoheptyl)-ε-dGuo lesion
in the 5′-TXG-3′ template (60).
The catalytic efficiency for dATP insertion opposite the 7-(2-
oxoheptyl)-ε-dGuo lesion of the 5′-TXG-3′ template was
16-40-fold higher than the other dNTPs. The mechanism can
either involve the instructive insertion of dATP opposite the
Thy situated on the 5′-side of the lesion through a dNTP-
stabilized misincoporation mechanism (35, 60, 61) or the

Bypass of 7-(2-Oxoheptyl)-ε-dGuo by Dpo4
Chem. Res. Toxicol., Vol. 23, No. 8, 2010 1339
Support for the dNTP-stabilized misincorporation mechanism
comes from the crystallographic analysis of the ternary complex
of Dpo4 bound to the 1,N2-ε-dGuo containing template paired
with a -1 primer, which showed dATP involved in nascent
Watson-Crick pairing with the 5′-Thy (35); a related structure
was also reported for the 5′-CXG-3′ template and dGTP. The
template strand was relatively unperturbed, and 1,N2-ε-Gua
provided an important π-stacking platform for the incoming
dNTP in both structures (60). The postinsertion complex of the
5′-TXG-3′ template showed that Ade at the primer terminus
was paired with 5′-Thy; once again, 1,N2-ε-Gua was well stacked
and stabilized the one-base deletion product. Similar crystal
structures were reported for the major malondaldehyde adduct
of dGuo (M1dGuo) bound to Dpo4 in the same sequence
contexts (57). The catalytic efficiency for dATP insertion
opposite 1, N2-ε-dGuo and M1dGuo by Dpo4 was 4.0 and 2.5
higher for the 5′-TXG-3′ template than the 5′-CXG-3′ template,
respectively; Km values that were 8- and 4-fold higher for 5′-
CXG-3′ were the major contribution to the differences. Insertion
of dGTP was most efficient for the 1,N2-ε-dGuo lesion in 5′-
CXG-3′. Insertion of dGTP opposite the 7-(2-oxoheptyl)-ε-dGuo
lesion in the 5′-CXG-3′ template was 4-fold less efficient than
misinsertion of dATP; however, the one-base deletion product
(5′-pTC-GTGA-3′) was nearly as abundant as the misinsertion
of Ade (5′-pTCAGTGA-3′), indicating that extension of the
misaligned primer with the Gua terminus paired with 5′-Cyt
was very favorable. These results support a dNTP-stabilized
misincoporation mechanism for the one-base deletion product
involving instructive insertion of the dNTP opposite the 5′-base
to the lesion (60).
This result is in contrast to the same duplex containing the 1,N2-
ε-Gua base, in which the Tm was 6 °C higher when opposite a
one-base deletion and 4 °C higher when paired with Ade as
compared to the 1,N2-ε-Gua·Cyt pair (Table 3). A full structure
of the 1,N2-ε-Gua·Ade duplex is currently in progress.
The catalytic efficiency for the insertion of dCTP, dTTP, and
dGTP opposite the 7-(2-oxoheptyl)-ε-dGuo lesion in the 5′-
TXG-3′ template (7) was 32-, 16-, and 41-fold lower than dATP
insertion, and this difference is reflected in the distribution of
the extension products. Insertion of dCTP opposite either etheno
lesion was the least efficient in both sequences, and the
5′-neighboring base had a minor influence in those relative
efficiencies. Interestingly, the error free extension product was
observed in 14% and 33% in the CXG-3′ template indicating
that extension from the ε-Gua·Cyt template-primer terminus
by Dpo4 was efficient in this sequence. A possible explanation
is that during translesion synthesis ε-Gua is involved in a
Hoogsteen pairing interaction with the complementary Cyt, as
observed for 1,N2-ε-dGuo in duplex DNA (38).
We recently reported the structure of the 1,N2-ε-dGuo lesion
in the same duplex, 5′-(CGCATXGAATCC)-3′·5′-(GGATTC-
CATGCG)-3′ (where X ) 1,N2-ε-dGuo). We established that
the 1,N2-ε-dGua base existed as a mixture of anti and syn
conformations about the glycosyl bond in the 5′-TXG-3′ (9)
sequence context at neutral pH (37, 38). Increasing the pH to
8.6 shifts the conformational equilibrium toward the anti
conformation (37), whereas decreasing the pH to 5.2 shifts the
equilibrium toward the syn conformation, accompanied by the
formation of a Hoogsteen base pair between 1,N2-ε-dGuo and
the complementary protonated dCyd (38). A similar conforma-
tional transition was observed for the 1,N2-ε-dGuo lesion in the
5′-CXC-3′ sequence context in DNA (64).
The NMR spectra of duplexes containing the 7-(2-oxoheptyl)-
ε-dGuo lesion were also dependent upon pH, indicative of a
pH-mediated conformational equilibrium. The likely explanation
is a syn-anti conformational equilibrium involving the glycosyl
bond, similar to that observed for 1,N2-ε-dGuo, although this
cannot be established with certainty. However, the pKa is
reduced as compared to the duplex containing the 1,N2-ε-dGuo
lesion, which exhibited a pKa value near neutrality. As detailed
in Table 3, the Tm of the 1,N2-ε-dGuo adduct in the same
sequence increased at pH 5.2 concomitant with the stabilization
of a Hoogsteen-like base pair under acidic conditions. In the
present case, the spectra are similar at pH 7 and pH 8.6 but
differ at pH 5.2, suggesting that the pKa of the transition is
shifted to the more acidic pH for the duplex containing the 7-(2-
oxoheptyl)-ε-dGuo lesion. This probably accounts for the
observation that the Tm of the 7-(2-oxoheptyl)-ε-dGuo containing
duplex remains constant over the range of pH 5.2 to pH 8.6.
Unfortunately, the 7-(2-oxoheptyl)-ε-dGuo lesion remains in a
disordered conformation throughout the pH range from pH
5.2-8.6. The structural disorder likely involves thermodynamic
destabilization attributed to the hydrophobic heptyl side chain
at the lesion site. This is consistent with thermal melting studies
showing that the 7-(2-oxoheptyl)-ε-dGuo containing duplex is
less stable than the duplex containing the 1,N2-ε-dGuo lesion.
The Streisinger slippage model represents a third possible
mechanism leading to the one-base deletion product. The
misalignment of the template and primer is caused by extrusion
of the modified base into an extra-helical loop, and this model
is often applied to deletions in repetitive sequence. NMR
structures of DNA duplexes containing C8-aromatic amines and
N2-BPDE-dGuo show that intercalation of the adduct moiety
into DNA stabilizes the slippage intermediate (62, 63). Crystal
structures of Dpo4 bound to DNA templates containing the
noncoding tetrahydrofuran model of an abasic site and the N2-
BPDE-dGuo adduct show that the modified nucleotide was part
of an extrahelical loop, which resulted in misalignment of the
template and primer (56, 59). In the case of C8-aromatic amines,
the intercalation of the adduct into the base stack stabilizes the
slippage intermediate when situated in a repeat sequence. This
feature was not present in the structure of Dpo4·BPDE-adduct
structures.
Nearly 90% of the extension products that were characterized
from the bypass of 7-(2-oxoheptyl)-ε-dGuo and 80% from the
1,N2-ε-dGuo lesion in the 5′-TXG-3′ template (7) were derived
from an initial insertion of dATP (one-base deletion or misin-
sertion of Ade). This result indicates that Dpo4 can efficiently
extend either the aligned or misaligned primer in which Ade
was opposite the lesion or 5′-Thy. Misinsertion of Ade was
preferred over the one-base deletion product (35% vs 24%) for
bypass of the 7-(2-oxoheptyl)-ε-dGuo lesion, whereas the
preference was reversed for the 1,N2-ε-dGuo lesion (16% vs
31%). This result may reflect disrupted stacking due to the
disordered conformation of the 7-(2-oxoheptyl)-ε-Gua base,
making the one-base deletion pathway less favorable. Interest-
ingly, the Tm values for the 7-(2-oxoheptyl)-ε-dGuo duplexes
(9) were insensitive to the nature of the complementary base
(Cyt, Ade, or a one-base deletion), suggesting that the 7-(2-
oxoheptyl)-ε-Gua base may be disordered in all three duplexes.
Conclusions
Oligonucleotides containing the 7-(2-oxoheptyl)-ε-dGuo le-
sion, an etheno adduct uniquely derived from lipid peroxidation,
have been synthesized using the phosphoramidite approach.
Translesion synthesis of the 7-(2-oxoheptyl)-ε-dGuo lesion by
Dpo4 in 5′-TXG-3′ (7) and 5′-CXG-3′ (8) local sequence
contexts was examined and compared to 1,N2-ε-dGuo. The

1340 Chem. Res. Toxicol., Vol. 23, No. 8, 2010
ChristoV et al.
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Formation of a substituted 1,N6-etheno-2′-deoxyadenosine adduct by
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extension products were sequenced by tandem mass spectrom-
etry using an improved protocol that enhances sensitivity. This
approach allowed us to identify extension products from the
bypass of the 1,N2-ε-dGuo lesion that were not previously
observed as well as revise the sequence of one of the extension
products. The major extension products for the 7-(2-oxoheptyl)-
ε-dGuo adduct was misinsertion of Ade followed by a one-
base deletion, whereas replication of the 1,N2-ε-dGuo lesion
resulted in more of the one-base deletion products. The 7-(2-
oxoheptyl)-ε-dGuo adduct was more miscoding than 1,N2-ε-
dGuo; replication of the lesions in the 5′-TXG-3′ (8) sequence
was more miscoding than in the 5′-CXG-3′ (7) sequence. The
one-base deletion product arises from either a dNTP-stabilized
misincoporation or a misincorporation-misalignment mecha-
nism and may be different depending on the lesion. NMR
analysis indicated that the structure of the 7-(2-oxoheptyl)-ε-
dGuo lesion in DNA is dependent upon pH. In the 5′-TXG-3′
(9) sequence context, it showed similar spectra at pH 7 and pH
8.6, as compared to pH 5.2, but the adduct remained disordered
across this pH range. The source of the pH-dependent confor-
mational transition remains to be determined but likely involves
interconversion between syn and anti conformations about the
glycosyl bond, similar to the duplex containing the 1,N2-ε-dGuo
lesion, but with a lower pKa value. This may contribute to the
differences in the translesion synthesis between the 7-(2-
oxoheptyl)-ε-dGuo and 1,N2-ε-dGuo lesions by Dpo4 polymerase.
Acknowledgment. This work was supported by NIH Grants
P01 ES05355 (to C.J.R. and M.P.S.) and R01 ES10375 (to
F.P.G.), and center grant P30 ES00267 (to C.J.R., M.P.S., and
F.P.G). Vanderbilt University, the Vanderbilt Center in Molec-
ular Toxicology (P30 ES00267), and the National Institute for
Research Resources (S10 RR005805) provided funding for the
NMR instrumentation. We also thank the reviewers for helpful
comments.
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Nakamura, H., Sawa, R., Matsui, S., and Matsuda, T. (2005) 4-Oxo-
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adduct formation in mouse organs. Ind. Health 43, 699–701.
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guanosine adducts by cis-2-butene-1,4-dial, a reactive metabolite of
furan. Chem. Res. Toxicol. 17, 1607–1613.
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(2006) Formation of 1,4-dioxo-2-butene-derived adducts of 2′-deoxy-
adenosine and 2′-deoxycytidine in oxidized DNA. Chem. Res. Toxicol.
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adduct of cis-2-butene-1,4-dial, a reactive metabolite of furan. Chem.
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Supporting Information Available: 1H, 13C, and 31P NMR
spectra, MS and CGE characterization of oligonucleotides,
sequences of oligonucleotides and primers, gels of extension
reactions, reconstructed ion profiles of the extension reactions,
CID mass spectra of extension products and standards, tables
of the observed and expected CID fragment ions for the
extension products, and calibration curves. This material is
available free of charge via the Internet at http://pubs.acs.
org.
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bioactivation of furan. Drug Metab. ReV. 38, 615–626.
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R-acetoxy-N-nitrosopiperidine with deoxyguanosine: Oxygen-depend-
ent formation of 4-oxo-2-pentenal and a 1,N2-ethenodeoxyguanosine
adduct. Chem. Res. Toxicol. 5, 706–712.
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