Site-Specific Incorporation of N-(2′-Deoxyguanosine-8-yl)-6-aminochrysene Adduct in DNA and Its Replication in Human Cells

Article abstract of DOI:10.1021/acs.chemrestox.0c00197

The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and a mammary carcinogen in rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. In mammalian cells, it is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields two major adducts with 2′-deoxyguanosine (dG), one at the C8-position, N-(dG-8-yl)-6-AC, and the other at the exocyclic N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total synthesis of a site-specific oligonucleotide containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC. Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG derivative served as the key reaction to furnish protected N-(dG-8-yl)-6-AC in 56% yield. The monomer for solid-phase DNA synthesis was prepared by its deprotection followed by conversion to the corresponding 5′-O-dimethoxytrityl 3′-phosphoramidite, which was used to synthesize a site-specifically adducted oligonucleotide. After purification and characterization, the adduct-containing oligonucleotide was incorporated into a plasmid and replicated in human embryonic kidney (HEK) 293T cells, which showed that N-(dG-8-yl)-6-AC stalls DNA replication as evidenced by 77% translesion synthesis (TLS) efficiency relative to the control and that the adduct is mutagenic (mutation frequency (MF) 17.8%) inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA polymerases in the bypass of N-(dG-8-yl)-6-AC using siRNA knockdown approach. TLS efficiency was reduced in hPol η-, hPol κ-, hPol ζ-, and hREV1-deficient HEK 293T cells to 66%, 45%, 37%, and 32%, respectively. Notably, TLS efficiency was reduced to 18% in cells with concurrent knockdown of hPol κ, hPol ζ, and REV1, suggesting that these three polymerases play critical roles in bypassing N-(dG-8-yl)-6-AC. MF increased to 23.1% and 32.2% in hPol κ- and hREV1-deficient cells, whereas it decreased to 11.8% in hPol ζ-deficient cells. This suggests that hPol κ and hREV1 are involved in error-free TLS of this lesion, whereas hPol ζ performs error-prone bypass.

Full text of DOI:10.1021/acs.chemrestox.0c00197

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Article
Site-specific incorporation of N-(2#-deoxyguanosine-8-yl)-6-
aminochrysene adduct in DNA and its replication in human cells
Paritosh Pande, Kimberly R. Rebello, Arindom Chatterjee, Spandana Naldiga, and Ashis K. Basu
Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.0c00197 • Publication Date (Web): 19 Jun 2020
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Site-specific incorporation of N-(2ʹ-deoxyguanosine-8-yl)-6-aminochrysene
adduct in DNA and its replication in human cells
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Paritosh Pande1, Kimberly R. Rebello1, Arindom Chatterjee, Spandana Naldiga, and Ashis
K. Basu*
Department of Chemistry, University of Connecticut, Storrs, CT 06269
1These two authors contributed equally to this work.
Keywords: nitroaromatic compounds, 6-aminochrysene, carcinogen-DNA adduct, translesion
synthesis, mutagenicity, cellular replication
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REV1

ee
hPol
ation
Error-fr
replic
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NH
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HEK 293T cells
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ABSTRACT
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The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and a mammary carcinogen in
rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. In mammalian cells, it is
metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways.
The nitroreduction pathway yields two major adducts with 2'-deoxyguanosine (dG), one at the C8-position,
N-(dG-8-yl)-6-AC, and the other at the exocyclic N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total
synthesis of a site-specific oligonucleotide containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC.
Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG
derivative served as the key reaction to furnish protected N-(dG-8-yl)-6-AC in 56% yield. The monomer
for solid-phase DNA synthesis was prepared by its deprotection followed by conversion to the
corresponding 5ʹ-O-dimethoxytrityl 3ʹ-phosphoramidite, which was used to synthesize a site-specifically
adducted oligonucleotide. After purification and characterization, the adduct-containing oligonucleotide
was incorporated into a plasmid and replicated in human embryonic kidney (HEK) 293T cells, which
showed that N-(dG-8-yl)-6-AC stalls DNA replication as evidenced by 77% translesion synthesis (TLS)
efficiency relative to the control and that the adduct is mutagenic (mutation frequency (MF) 17.8%)
inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA
polymerases in the bypass of N-(dG-8-yl)-6-AC using siRNA knockdown approach. TLS efficiency was
reduced in hPol η-, hPol κ-, hPol ζ-, and hREV1-deficient HEK 293T cells to 66%, 45%, 37%, and 32%,
respectively. Notably, TLS efficiency was reduced to 18% in cells with concurrent knockdown of hPol κ,
hPol ζ, and REV1, suggesting that these three polymerases play critical roles in bypassing N-(dG-8-yl)-6-
AC. MF increased to 23.1% and 32.2% in hPol κ- and hREV1-deficient cells, whereas it decreased to 11.8%
in hPol ζ-deficient cells. This suggests that hPol κ and hREV1 are involved in error-free TLS of this lesion,
whereas hPol ζ performs error-prone bypass.
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INTRODUCTION
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Nitropolycyclic aromatic hydrocarbons (NO2-PAHs) are common environmental pollutants as they are
formed during combustion of diesel and other fossil fuels.1-4 They are also present in certain foods and
beverages. The International Agency for Research on Cancer (IARC) labeled diesel exhaust as
“carcinogenic to humans” (Group 1) and one of its NO2-PAH contaminant 6-nitrochrysene (6-NC) as
“probably carcinogenic to humans” (Group 2A).5 6-NC is mutagenic in bacteria and carcinogenic in
experimental animals.6-8 It is the most potent carcinogen ever tested in the newborn mouse assay and its
carcinogenic potency in rat mammary gland is higher than that of the potent carcinogens benzo[a]pyrene
and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP).7-11
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NO2
6-NC
Nitroreduction
Ring Oxidation
OH
OH
HN OH
NO2
N-Hydroxy-6-AC
1,2-DHD-6-NC
OH
OH
O
N
O
N
N
N
N
NH
NH
H
N
NH
NH2
NH2
N
NH2
1,2-DHD-6-AC
OH
Adduct II
Adduct I
OH
O
N
N
N
H
O
NH
N
NH2
1,2-DHD-6-AC-3,4-Epoxide
Adduct III
DNA Adducts
Scheme 1. Metabolic activation of 6-NC that leads to DNA adduct formation. Adduct I = N-(dG-8-yl)-6-
AC, adduct II = 5-(dG-N2-yl)-6-AC, and adduct III = N-(dI-8-yl)-6-AC.
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6-NC is metabolically activated by two different pathways: the first involves nitroreduction to form
6-hydroxyaminochrysene (N-OH-6-AC) and the second involves both nitroreduction and ring oxidation
(Scheme 1).6,12-14 N-OH-6-AC, formed in the first pathway forms three DNA adducts, N-(dG-8-yl)-6-AC,
5-(dG-N2-yl)-6-AC, and N-(dI-8-yl)-6-AC, the latter being generated from the 2ʹ-deoxyadenosine adduct,
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N-(dA-8-yl)-6-AC.15
The
second
pathway
generates
trans-1,2-dihydroxy-1,2-dihydro-6-
hydroxyaminochrysene (1,2-DHD-6-NHOH-C), which forms 5-(dG-N2-yl)-1,2-DHD-6-AC, N-(dG-8-yl)-
1,2-DHD-6-AC, and N-(dA-8-yl)1,2-DHD-6-AC. All these DNA adducts have been detected in several
organs of rats treated with 6-NC orally or by intraperitoneal injection.16
There are no reports to synthesize the 6-NC-DNA adducts by a total synthetic approach, except we
recently published an efficient method to prepare N-(dA-8-yl)-6-AC-containing oligonucleotide.17
However, El-Bayoumy and coworkers have synthesized oligonucleotides containing N-(dG-8-yl)-6-AC, N-
(dG-8-yl)-1,2-DHD-6-AC, and N-(dA-8-yl)-6-AC by reacting N-OH-6-AC or 1,2-DHD-6-NHOH-C with
oligonucleotides containing either a single dG or dA, as appropriate, to prepare small amount of the
adducted oligonucleotides.18 They also investigated repair of these adducts in human cells.18 When the
nucleotide excision repair (NER) efficiency of N-(dG-8-yl)-1,2-DHD-6-AC, N-(dG-8-yl)-6-AC, and N-
(dG-8-yl)-6-AC were compared in HeLa cell extracts, N-(dG-8-yl)-6-AC repair was most facile. NER of
N-(dG-8-yl)-1,2-DHD-6-AC is ~2-fold more resistant than N-(dG-8-yl)-6-AC whereas N-(dA-8-yl)-6-AC
is repaired 8-fold more slowly than N-(dG-8-yl)-6-AC. However, replicative properties of these DNA
adducts in mammalian cells have not been investigated.
As indicated earlier, we have recently developed a total synthesis method to prepare site-
specifically incorporated N-(dA-8-yl)-6-AC and established that this adduct is mutagenic in Escherichia
coli.17 Herein, we report the total synthesis approach of N-(dG-8-yl)-6-AC in an oligonucleotide, which was
used to investigate the mutagenicity of this adduct in human embryonic kidney (HEK) 293T cells. We also
determined the roles of the translesion synthesis (TLS) DNA polymerases on the bypass and mutagenicity
of N-(dG-8-yl)-6-AC.
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MATERIALS AND METHODS
Materials
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All materials, including reagents and solvents, were of commercial grade. [γ- P] ATP was purchased from
Perkin Elmer Health Sciences Inc. (Shelton, CT). The enzymes were purchased from New England Biolabs
(Beverly, MA). All unmodified oligodeoxynucleotides were obtained from Integrated DNA Technologies
(Coralville, IA). Synthetic siRNA duplex against Pol H (SI02663619), Pol K (SI04930884), Pol I
(SI03033310), hREV1 (SI00115311), and negative control siRNA (1027280) were purchased from Qiagen
(Valencia, CA), whereas the same for Rev3 was obtained from Integrated DNA Technologies (Coralville,
IA). Sequences of all siRNAs have been reported.21 NMR spectra were recorded on a Bruker Advance 400
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MHz instrument. The H and C NMR data were recorded to the internal deuterated solvent relative to
TMS at 0 ppm. High resolution mass spectroscopy (HRMS) was carried out on a QStar Elite
electrospray/nanospray quadrupole time-of-flight mass spectrometer (AB Sciex) in positive ionization
mode, and the data were analyzed using MassLynx. Flash chromatography was performed on a Teledyne
ISCO CombiFlash Rf automated MPLC system with silica or aluminum oxide columns.
Methods
O6-Benzyl-8-bromo-3',5'-O-bis(tert-butyldimethylsilyl)-N2-dimethoxytrityl-2'-deoxyguanosine, 3, was
synthesiszed from 2'-deoxyguanosine, 1, as reported.19 For introduction of the bromine at the C8 position
of 2'-deoxyguanosine, freshly recrystallized NBS was used. In the Mitsunobu reaction to protect the
carbonyl moiety, DIAD was used in place of DEAD, which gave better yield of the product.
8N-(6-Aminochrysene)-O6-benzyl-3',5'-O-bis(tert-butyldimethylsilyl)-N2-dimethoxytrityl-2'-
deoxyguanosine (4). O6-Benzyl-8-bromo-3',5'-O-bis(tert-butyldimethylsilyl)-N2-dimethoxytrityl-2'-
deoxyguanosine, 3, (5.09 g, 5.26 mmol), 6-aminochrysene (1.92 g, 7.89 mmol),
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tris(dibenzylideneacetone)dipalladium
(Pd2(dba)3)
(0.19
g,
0.21
mmol)
and
rac-2,2'-
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bis(diphenylphosphino)-1,1'-binaphthyl (BINAP, 0.38 g, 0.61 mmol) were suspended in 20 mL dry toluene.
The solution was degassed by purging argon for 45 minutes and then heated under argon at 90°C for an
hour. Next, NatBuO (0.76 g, 7.90 mmol) was added to the reaction and stirred at 100°C for another hour.
The reaction was cooled, diluted with 50 mL ether and filtered through celite. The filtrate was evaporated
and purified by aluminum oxide column chromatography with a step gradient of EtOAc (0-12%) in hexane
as the mobile phase. The product was isolated as a dark green solid (3.22 g, yield of 56%).
1H NMR (400 MHz, CDCl3) δ (ppm) 9.56 (s, 1H, N8-H), 8.85 – 8.78 (m, 2H, CR-H2), 8.66 (d, J = 9.1 Hz,
1H, CR-H), 8.40 (s, 1H, CR-H), 8.13 -8.11 (m, 1H, CR-H) 7.98 -7.91 (m, 2 H, CR-H), 7.73-7.71 (m, 1H,
CR-H), 7.69-7.67 (m, 1H, CR-H), 7.62 – 7.60 (m, 2H, CR-H), 7.42 –7.24 (m, 16H, CR-H4 + Bz-H5 + DMT-
H9), 6.82 – 6.79 (m, 4H, DMT-H4), 6.23 (br, 1H, C1'-H), 6.13 (s, 1H, N2-H), 5.11 (br, 2H, Bz-CH2), 4.53
(br-s, 1H, C4’-H), 4.16 (m, 1H, C3’-H), 3.90 -3.86 (br-m, 2H, C5'-H2) 3.81 (s, 6H, 2 X DMT-OMe), 2.90 (br-
m, 1H, C2’-H), 2.28 -2.26 (br-m, 1H, C2’-H), 0.96 (s, 9H, tBu-H), 0.66 (s, 9H, tBu-H), 0.14 (s, 6H, 2 X
CH3), -0.16 (s, 3H, CH3), -0.24 (s, 3H, CH3) (Figure S1.1 in SI).
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13C NMR (101 MHz, CDCl3) δ 157.9, 155.9, 153.28, 148.5, 146.32, 138.68, 138.52, 137.21, 133.78, 132.36,
131.44, 130.41, 130.25, 130.19, 129.08, 129.02, 128.37, 128.19, 128.11, 127.81, 127.50, 127.42, 126.85,
126.43, 126.35, 126.25, 125.83, 125.11, 123.99, 123.84, 121.38, 120.87, 112.79, 112.76, 112.20, 88.20,
85.58, 72.58, 70.11, 67.54, 63.14, 55.12, 39.50, 36.65, 31.56, 26.89, 25.80, 25.65, 25.26, 20.68, 18.21,
17.98, -4.59, -4.75, -5.71, -5.78 (Figure S1.2 in SI).
ESI-MS (positive mode): m/z calcd for C68H76N6O6Si2.H+ 1129.5, observed 1129.9 Da [M + H]+ (Figure
S2.1 in SI).
8N-(6-Aminochrysene)-O6-benzyl-3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxyguanosine. 8N-
(6-Aminochrysene)-O6-benzyl-3',5'-O-bis(tert-butyldimethylsilyl)-N2-dimethoxytrityl-2'-deoxyguanosine,
4, (0.45 g, 0.40 mmol) was reacted with 0.01M methanolic HCl (4 mL, 0.04 mmol) and stirred for 12 h.
After the reaction was complete, solvent was evaporated and purified by silica gel column chromatography
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with a step gradient of methanol (0-2%) in DCM as the mobile phase. The product was isolated as a yellow
solid (1.42 g, yield of 81%).
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1H NMR (400 MHz, Chloroform-d) δ 9.55 (s, 1H, N8-H), 8.85 – 8.80 (m, 2H, CR-H2), 8.65-8.63 (d, J = 9.1
Hz, 1H, CR-H), 8.40 (m, 1H, CR-H), 8.18 – 8.16 (m, 1H, CR-H), 7.99 – 7.97 (m, 1H, CR-H), 7.93 – 7.90
(m, 1H, CR-H), 7.74 – 7.34 (m, 9H, CR-H4 + Bz-H5), 6.45 (dd, J = 7.8, 5.5 Hz, 1H, C1'-H), 5.69 (q, 2H,
Bz-CH2), 4.89 (s, 2H, N2-H), 4.68 (m, 1H, C3’-H), 4.20 (ddd, J = 3.3 Hz, 1H, C4’-H), 4.01 (dd, J = 5.0, 3.7
Hz, 1H, C5’-H), 3.87 (dd, J = 4.9 Hz, 3.5 Hz, 1H, C5’-H), 3.22 (ddd, 1H, C2’-H), 2.44 -2.39 (m, 1H, C2’-H),
1.02 (s, 9H, tBu-H), 0.69 (s, 9H, tBu-H), 0.21 (d, J = 3.0 Hz, 6H, 2 X CH3), -0.13 (s, 3H, CH3), -0.22 (s,
3H, CH3) (Figure S1.3 in SI).
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13C NMR (101 MHz, CDCl3) δ 158.30, 157.28, 153.83, 148.51, 137.06, 133.81, 132.28, 131.32, 130.29,
128.86, 128.16, 127.87, 127.56, 126.77, 126.37, 126.16, 125.80, 125.12, 123.75, 123.71, 121.41, 120.78,
113.00, 112.72, 87.85, 85.06, 72.28, 67.96, 62.95, 39.08, 34.58, 31.50, 25.72. 25.55, 22.56, 18.13, 17.92, -
4.64, -4.81, -5.80, -5.86 (Figure S1.4 in SI).
ESI-MS (positive mode): m/z calcd for C47H58N6O4Si2.H+ 827.4, observed 827.7 Da [M + H]+ (Figure S2.2
in SI).
8N-(6-Aminochrysene)- 3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxyguanosine (5). 8N-(6-
Aminochrysene)-O6-benzyl-3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxyguanosine (0.70 g, 0.85 mmol)
was dissolved in 20 mL dry methanol. 10% Pd -black (90 mg) was added to it and the solution was stirred
overnight under hydrogen. The solvent was evaporated and the reaction mixture was dried and purified by
silica gel column chromatography with a step gradient of methanol (0-6%) in DCM as the mobile phase.
The product was isolated as a white solid (0.55 g, yield of 87%).
1H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H, N8-H), 8.98 (d, J = 8.4 Hz, 1H), 8.82 (d, J = 9.1 Hz, 1H,
CR-H), 8.67 (s, 1H, CR-H), 8.56 (d, J = 8.3 Hz, 1H, CR-H), 8.44 (s, 1H, CR-H), 8.28 (dd, J = 8.4, 1.4 Hz,
1H, CR-H), 8.07 -8.04 (m, 1H, CR-H), 7.98 (d, J = 9.1 Hz, 1H, CR-H), 7.81 – 7.63 (m, 4H, CR-H4), 6.30
(s, 2H, N2-H), 6.26 -6.23 (dd, J = 8.2, 6.1 Hz, 1H, C1'-H), 4.41 (ddd, J = 5.4, 2.4 Hz, 1H, C3’-H), 3.79 – 3.71
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(m, 2H, C4’-H + C5’-H), 3.64- 3.61 (m C5’-H), 3.29 -3.21 (m, 1H, C2’-H), 2.12 -2.04 (m, 1H, C2’-H), 0.73
(s, 9H, tBu-H), 0.69 (s, 9H, tBu-H), -0.04 (s, 3H, CH3), -0.09 (s, 3H, CH3), -0.16 (s, 6H, 2 X CH3) (Figure
S1.5 in SI).
9
13C NMR (101 MHz, DMSO-d6) δ 155.87, 152.70, 150.60, 144.66, 137.74, 132.06, 131.01, 129.20, 128.42,
128.18, 127.08, 126.53, 126.32, 126.05, 125.33, 124.14, 123.90, 122.82, 121.33, 113.77, 108.95, 87.19,
83.40, 72.77, 63.14, 36.77, 25.76, 25.63, 25.48, 17.88, 17.50, -5.02, -5.05, -5.62, -5.67 (Figure S1.6 in SI).
ESI-MS (positive mode): m/z calcd for C40H52N6O4Si2.H+ 737.5, observed 737.4 Da [M + H]+ (Figure S2.3
in SI).
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8N-(6-Aminochrysene)-3',5'-O-bis(tert-butyldimethylsilyl)-N2-DMF-2'-deoxyguanosine. 8N-
(6-Aminochrysene)-3',5'-O-bis(tert-butyldimethylsilyl)-2'-deoxyguanosine (0.50 gm, 0.68 mmol) was
dissolved in 10 mL methanol and N, N-dimethylformamide dimethylacetal (0.36 mL, 2.72 mmol) was
dropwise added to the solution and stirred at room temperature for 24 h. After the reaction was complete,
solvent was evaporated and the product was purified by silica gel column chromatography with a step
gradient of methanol (0-5%) in DCM as the mobile phase. The product was isolated as a white solid (0.54
g, yield of 99%).
1H NMR (300 MHz, Chloroform-d) δ 9.25 (s, 1H, N8-H), 8.82 -8.78 (m, 2H, CR-H2), 8.71 (brs, 1H), 8.65
(d, J = 9.17 Hz, 1H, CR-H), 8.59 (s, 1H, CR-H), 8.12 – 8.10 (m, 2H, CR-H2), 7.96 – 7.90 (m, 2H, CR-H2),
7.71 -7.57 (m, 4H, CR-H4), 6.56 -6.51 (m, 1H, C1'-H), 4.53 -4.51 (m, 1H, C3’-H), 4.15 -4.14 (m, 1H, C4’-
H), 3.99 -3.94 (m, 1H, C5’-H), 3.89 -3.84 (m, 1H, C5’-H), 3.15 (s, 3H, DMF-CH3), 3.09 (s, 3H, DMF-CH3),
2.89 -2.79 (m, 1H), 2.34 -2.28 (m, 1H), 0.93 (s, 9H, tBu-H), 0.59 (s, 9H, tBu-H), 0.10 (s, 6H, 2 X CH3), -
0.23 (s, 3H, CH3), -0.34 (s, 3H, CH3) (Figure S1.7 in SI).
13C NMR (101 MHz, Chloroform-d) δ 157.45, 156.94, 154.97, 149.07, 147.44, 134.12, 132.30, 131.48,
130.33, 129.02, 128.11, 126.42, 126.39, 126.26, 126.13, 125.90, 125.31, 124.13, 123.72, 121.92, 120.85,
116.99, 113.84, 88.02, 84.30, 72.68, 63.26, 41.14, 39.82, 35.02, 25.72, 25.63, 18.25, 17.92, -4.62, -4.75, -
5.73, -5.81 (Figure S1.8 in SI).
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ESI-MS (positive mode): m/z calcd for C40H52N6O4Si2.H+ 792.4, observed 792.6 Da [M + H]+ (Figure S2.4
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6
in SI).
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8N-(6-Aminochrysene)-N2-DMF-2'-deoxyguanosine. 8N-(6-Aminochrysene)-3',5'-O-bis(tert-
butyldimethylsilyl)-N2-DMF-2'-deoxyguanosine (0.483 g, 0.609 mmol) was dissolved in 10 mL dry THF,
and a solution of Et3N.3HF (0.500 mL, 3.07 mmol) was added. The reaction mixture turned red
immediately. The reaction was stirred for 12 h and monitored by TLC (92/8 CHCl3/MeOH v/v). The
solvents were removed under reduced pressure, and the resulting yellow solid was purified by silica gel
column chromatography with a step gradient of methanol (0-10%) in DCM as the mobile phase. The
product was isolated as a red solid (0.275 g, yield of 80 %).
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1
1H NMR (400 MHz, DMSO-d6) H NMR (400 MHz, DMSO-d6) δ 11.24 (s, 1H, N8-H), 9.02 – 8.97 (m,
2H, DMF-H + CR-H), 8.86 (d, J = 9.1 Hz, 1H, CR-H), 8.73 -8.70 (m, 2H, CR-H2), 8.56 (s, 1H, CR-H),
8.19 – 8.17 (m, 1H, CR-H), 8.10 – 8.03 (m, 2H, CR-H2), 7.80 – 7.68 (m, 3H, CR-H3), 6.51 (dd, J = 8.6, 6.3
Hz, 1H, C1'-H), 5.44 (brs, 1H, 5’-OH), 5.33 (brs, 1H, 3’-OH), 4.51 (m, 1H, C3’-H), 3.94 (m, 1H, C4’-H),
3.81 -3.79 (m, 1H, C5’-H), 3.69 (m, 1H, C5’-H), 3.17 (s, 3H, DMF-CH3), 3.04 (s, 4H, DMF-CH3 + C2’-H),
2.23 -2.21 (m, 1H, C2’-H) (Figure S1.9 in SI).
13C NMR (101 MHz,DMSO-d6) δ 157.57, 156.54, 155.88, 148.74, 146.64, 136.39, 131.97, 130.98, 129.39,
128.44, 128.05, 127.40, 127.00, 126.71, 126.63, 126.34, 125.97, 125.00, 123.76, 123.56, 123.23, 123.33,
113.05, 87.39, 83.24, 71.00, 61.38, 57.50, 40.57, 34.56, 19.17, 13.45 (Figure S1.10 in SI).
ESI-MS (positive mode): m/z calcd for C31H29N7O4.H+ 564.3, observed 564.3 Da [M + H]+ (Figure S2.5 in
SI).
8N-(6-Aminochrysene)-N2-DMF-5ʹ-O-(4,4ʹ-dimethoxytrityl)-2'-deoxyguanosine.
8N-(6-
Aminochrysene)-N2-DMF-2'-deoxyguanosine (0.10 g, 0.18 mmol) was extensively dried by evaporating
with pyridine three times. It was then combined with DMT-Cl (92 mg, 0.27 mmol) and dissolved in 5 mL
dry pyridine. The reaction was stirred for 6 h at room temperature and monitored by TLC (95/4/1
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CHCl3/MeOH/NEt3 v/v). The solvent was removed under reduced pressure, and the resulting yellow solid
was purified by silica gel column chromatography with a step gradient of methanol (0-5%) in DCM
containing 1% NEt3 as the mobile phase. The product was isolated as a yellow solid (0.14 mg, yield of
88%).
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1H NMR (400 MHz, CD2Cl2) δ 9.42 (brs, 1H, N8-H), 8.74 (d, J = 8.3 Hz, 1H, CR-H), 8.68 (s, 1H, CR-H),
8.60 (d, J = 9.1 Hz, 1H, CR-H), 8.49 (s, 1H, CR-H), 8.04 (d, J = 8.1 Hz, 1H, CR-H), 7.97 -7.95 (m, 1H,
CR-H), 7.89 -7.88 (m, 2H-CR-H2), 7.66 -7.62 (m, 1H), 7.55 -7.51 (m, 1H, CR-H), 7.48 -7.41 (m, 2H), 7.28
-7.27 (m, 2H), 7.12 -7.05 (m, 4H), 7.01 -7.00 (m, 3H), 6.67 -6.66 (m, 1H, C1'-H), 6.38 -6.34 (m, 4H), 5.05
-5.03 (brs, 1H, 5’-OH), 4.67 (brs, 1H, 3’-OH), 4.30 - 4.25 (m, 1H, C3’-H), 3.72 – 3.65 (m, 1H), 3.59 -3.57
(m, 1H, C4’-H), 3.43 -3.41 (m, 6H), 3.57 -3.03 (m, 1H), 3.10 (s, 3H DMF-CH3), 3.02 -2.98 (s, 4H, DMF-
CH3 + C2’-H), 2.35 -2.25 (m, 1H, C2’-H) (Figure S1.11 in SI).
13C NMR (101 MHz, CD2Cl2) δ 158.80, 158.75, 158.66, 157.89, 155.93, 150.27, 149.09, 144.35, 135.65,
134.95,132.62, 131.71, 130.99, 130.22, 129.01, 128.84, 128.76, 128.69, 128.10, 127.21, 126.99, 126.91,
126.35, 123.96, 123.91, 123.68, 121.21, 116.63, 116.09, 113.23, 86.99, 86.88, 84.87, 71.82, 63.99, 55.29,
54.39, 53.19, 41.78, 39.73, 35.37, 23.72, 20.31, 15.11, 13.74 13.56, 7.74.
ESI-MS (positive mode): m/z calcd for C52H47N7O6.H+ 866.7, observed 866.2 Da [M + H]+ (Figure S2.6
in SI).
8N-(6-Aminochrysene)-N2-DMF-5ʹ-O-(4,4ʹ-dimethoxytrityl)-3ʹ-cyanoethyl-2ʹ-
deoxyguanosine phosphoramidite (6). 8N-(6-Aminochrysene)-N2-DMF-5’-O-(4,4'-dimethoxytrityl)-2'-
deoxyguanosine (50 mg, 0.058 mmol) was co-evaporated twice with 5 mL dry dichloromethane, vacuum
dried overnight, and then dissolved in 3 mL DCM. Anhydrous diisopropylethylamine (0.021 mL, 0.12
mmol) was added to the solution and then 2-cyanoethyl-N, Nʹ-diisopropylchlorophosphoramidite (0.014
mL, 0.06 mmol) was added dropwise. After 15 minutes, more DIEA (0.021 mL, 0.12 mmol) was added.
The reaction mixture was stirred for another 15 minutes. Thereafter, the mixture was cooled in an ice bath
and methanol (1 mL) was added. The work-up involved extraction of the solution with 20 mL DCM (2×),
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followed by washing of the DCM layer with 10 mL sodium bicarbonate (2×) and 10 mL saturated sodium
chloride (1×). The organic layer was dried with anhydrous sodium sulfate and the solvent was removed
under reduced pressure. The resulting yellow paste was purified by silica gel column chromatography with
a step gradient of methanol (0-1%) in DCM containing 1% NEt3 as the mobile phase. The product was
isolated as a pale greenish yellow solid (41.4 mg, yield of 68%).
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1H NMR (400 MHz, CD2Cl2, two diastereomers) δ 8.79 (d, J = 7.8 Hz, 1H), 8.63 (d, J = 8.04 Hz, 1H), 8.59
(s, 1H), 8.45 (d, J = 13.5 Hz, 1H), 8.05 -8.00 (m, 2H), 7.92 -7.89 (m, 3H), 7.71 -7.68 (m, 1H), 7.54 -7.44
(m, 3H), 7.29 (s, 2H), 7.15 -7.04 (m, 8H), 6.57 (m, 1H), 6.42 – 6.39 (m, 4H), 4.85 (m, 1H), 4.32 – 4.28 (m,
1H), 3.72 -3.69 (m, 2H), 3.58 -3.55 (m, 2H), 3.45 -3.44 (m, 7H), 3.39 -3.37 (m, 2H), 3.11 (m, 4H), 3.04 (s,
3H), 2.52 -2.48 (m, 1H), 2.38 -2.35 (m, 1H), 1.16 -1.11 (m, 12H) (Figure S1.12 in SI).
13C NMR (101 MHz, CD2Cl2) δ 158.92,158.88, 156.15, 149.98, 144.15, 135.45, 132.66, 131.77, 130.68,
130.63, 130.43, 130.26, 130.18, 129.04, 128.90, 128.86, 128.77, 128.69, 128.59, 128.39, 128.19, 127.39,
127.26, 126.96, 124.03, 123.96, 123.54, 121.26, 113.72, 113.30, 87.63, 87.10, 85.84, 85.57, 84.85, 84.28,
75.03, 74.18, 73.99, 63.89, 63.20, 63.04, 58.99, 58.91, 58.80, 58.72, 57.63, 55.63, 55.34, 55. 31, 54.39,
53.15, 43.73, 41.67, 35.40, 34.62, 24.79, 23.36, 21.88, 20.82, 15.10, 7.68.
31P NMR (162 MHz, CDCl3) δ 149.06, 148.56 (Figure S1.13 in SI).
ESI-MS (positive mode): m/z calcd for C61H64N9O7P.H+ 1066.7, observed 1066.2 Da [M + H]+ (Figure
S2.7 in SI).
Synthesis of oligonucleotide using modified nucleoside 8N-(6-aminochrysene)-N2-DMF-5ʹ-O-
(4,4ʹ-dimethoxytrityl)-3ʹ-cyanoethyl-2ʹ-deoxyguanosine phosphoramidite monomer via solid-state
oligonucleotide synthesis. A conventional DNA synthesizer equipped with the phosphoramidite along with
the required reagents was used to synthesize the oligonuclotide 5ʹ-GTG CG*T GTT TGT-3ʹ, where G*
represents N-(dG-8-yl)-6-AC. The oligonuclotide was synthesized in DMT-off mode, where the final 5ʹ-
DMT group was cleaved before it was released from solid support. The oligomer was then deprotected
using standard conditions and finally 0.25M 2-mercaptoethanol was added into it to avoid oxidative
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degradation and lypholilized. This crude oligonucleotide was then purified in RP-HPLC using C-18
semiprep column (Phenomenex Gemini-NX-5µ) with an eluent of 0.1 M NH4OAc/ 1% CH3CN-H2O
(Solvent A) and 80 % CH3CN/20% H2O (solvent B) with a linear gradient (0 to 40 minutes, 2% to 40 % )
at a flow rate of 4 mL/min monitored at both 254 nm and 350 nm. Purified oligomers were collected in 1.5
mL microcentrifuge tubes, filtered and washed through amicon 3K ultracel filters to get rid of the excess
salt. Desalted fractions were then dried using a speedvac at 40 ° C and characterized with HRMS in negative
mode; 12 mer: 5ʹ-GTG CG*T GTT TGT-3ʹ HRMS calcd: 3939.7, found 3939.0 (Figure S2.8 in SI). Finally
the synthetic yield was quantified using UV-measurements.
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The purity of the oligonucleotide was further checked by comparing against a control sequence 5ʹ-
GTG CGT GTT TGT-3ʹ by 20% polyacrylamide urea deneaturing gel electrophoresis with [γ-32P]ATP
labelling.
Construction and characterization of a pMS2 vector containing a single N-(dG-8-yl)-6-AC
adduct and its replication in HEK 293T cells. We have constructed a single-stranded pMS2 vector
containing a single N-(dG-8-yl)-6-AC adducted site. This vector contained neomycin and ampicillin
resistance genes and is prepared as previously reported.20,21 The NC and various siRNA knockdown HEK
293T cells were grown to ∼90% confluency and transfected with 150 ng of construct in 6 µl of
Lipofectamine cationic lipid reagent (Invitrogen, Carlsbad, CA). Following transfection with modified or
◦
unmodified pMS2 vector, the cells were allowed to grow at 37 C in 5% CO2 for 48 h and the plasmid DNA
was collected and purified. It was used to transform E. coli (DH10B), and transformants were analyzed by
oligonucleotide hybridization followed by DNA sequence analysis.20,21
TLS assay in human cells. The lesion-containing or control pMS2 vector was mixed with an equal
amount of a single-stranded pMS2 DNA vector (internal control), which contained a 12-mer
oligonucleotide sequence different from the N-(dG-8-yl)-6-AC containing sequence (or control) DNA
sequence. The unmodified vector was used as an internal control and gave equal number of progeny as the
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control vector. TLS efficiency was determined as the percentages of the colonies originating from the N-
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6
(dA-8-yl)-6-A containing plasmid relative to the internal control.
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Mutational analyses of TLS products from human cells with polymerases knockdowns. For
siRNA knockdown experiments, prior to transfection of the control and N-(dG-8-yl)-6-AC containing
vectors, synthetic siRNA duplex was transfected into HEK 293T cells using Lipofectamine. HEK 293T
cells were plated in 6-well plates at 50% confluence. After 24 h incubation, they were transfected with 100
pmoles of siRNA duplex mixed with Lipofectamine, diluted in Opti-MEM (Gibco), per well. One day
before transfection of the plasmid, cells were seeded in 24-well plates at 70% confluence. Cells were then
co-transfected with another aliquot of siRNA and either control plasmid or lesion-containing plasmid. After
24 h incubation, progeny plasmids were isolated as described earlier.21
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RESULTS
Synthesis of an oligonucleotide containing N-(dG-8-yl)-6-AC. A key step in the synthesis of the protected
monomer of N-(dG-8-yl)-6-AC was the Buchwald-Hartwig coupling, which typically works best if the
primary amine at the 2 position of dG is protected with a DMT group.17,22,23 This has been attributed to the
increased solubility of dG in the reaction with the non-polar DMT group. Protected Br-dG, including a
DMT group on the exocyclic amine at the 2-position, 3, was prepared by a slight modification of reported
methods. Preliminary evaluation of different Pd catalysts, base, solvents and conditions indicated that
Pd2(dba)3 with BINAP and NaOtBu worked better than Pd(OAC)2 with BINAP and Cs2CO3 in the coupling
reactions of protected Br-dG with 6-aminochrysenene, which was also observed in the case of C8-dG-3-
aminobenzanthrone synthesis. Accordingly, Buchwald-Hartwig coupling of 3 with 6-aminochrysene was
carried out with Pd2(dba)3 and BINAP in toluene at 90°C for an hour followed by NaOtBu base treatment
for an additional hour at 100°C. The coupled product, 4, was obtained in 56% yield. It is noteworthy that
when coupling reactions were carried out for longer periods, the product was partially degraded instead of
providing a higher yield of the coupled product.
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Scheme 2. Synthesis of 5ʹ-DMT-protected 3ʹ-phosphoramidite 6a
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9
O
OBn
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N
NH
NH2
N
N
N
Br
a, b, c
N
N
HO
N
NH2
TBDMSO
O
O
HO
TBDMSO
1
2
OBn
N
N
N
Br
DMT
N
N
d
H2N
TBDMSO
O
H
e
TBDMSO
3
OBn
N
O
N
f, g
N
N
N
N
HN
NH
NH2
HN
DMT
N
N
TBDMSO
O
TBDMSO
H
O
4
TBDMSO
TBDMSO
5
O
N
h, i, j, k
N
NH
HN
N
DMT-O
N
N
O
O
P
NC
O
N
6
aReagents and conditions: (a) NBS, CH3CN, 1 h, rt, 89%; (b) TBDMSCl, imidazole, DMS, 12 h, rt, 94%;
(c) PPh3, PhCH2OH, DIAD, dioxane, 12 h, 78%; (d) DMT-Cl, DMAP, pyridine, 12 h, rt, 91%; (e) 6-
aminochrysene, Pd2(dba)3, BINAP, NaotBu, 90 oC, 1h, 56%; (f) HCl, methanol, 81%; (g) 10% Pd-C, H2,
87%; (h) DMF dimethylacetal, methanol, 24h, rt, 99%; (i) Et3N.3HF, dry THF, 12h, rt, 80%; (j) DMT-Cl,
DMAP, pyridine, 6h, rt, 88%; (k) DIEA, CH2Cl2, 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite,1h,
rt, 68%.
We determined, however, that all successive products after the Buchwald-Hartwig coupling are
extremely prone to degradation, as they are highly acid-, light-, temperature-, and pressure-sensitive. The
reaction mixtures were wrapped in aluminum foil to prevent them from undergoing photodecomposition.
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Also, if the synthesis was paused at any point, the product was stored at -20 oC to prevent degradation. The
5
6
phosphoramidite product, 6, was wrapped in aluminum foil and stored at -80 oC.
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The Buchwald-Hartwig coupling product 4 was purified via aluminum oxide column
chromatography. The DMT group was cleaved with a very dilute acid (0.01M methanolic HCl) and the
O6-benzyl was removed with 10% Pd-black and H2 to give 5. Subsequently, the deprotection of the TBDMS
groups was carried out with 1M TBAF in THF. Exocyclic amine at the 2-position of the product was
protected with a DMF group with N, N-dimethylformamide dimethyl acetal. For the DMT protection of the
5'-OH group, the reaction mixture was co-evaporated with pyridine three times to remove any moisture.
Likewise, for the final step to incorporate the phosphoramidite at the 5'-OH, the 5'-DMT-protected
monomer was dried overnight under vacuum, to ensure no moisture would be present in the reaction
mixture. The phosphoramidite reaction was performed in a glovebag under an argon atmosphere. The final
two reaction products were purified on a basified silica gel column to prevent any degradation of the
products by the acidity of the silica gel. Following spectroscopic characterization of the synthesized
phosphoramidite adduct, 6, it was used for incorporation into an oligonucleotide via solid-phase
oligonucleotide synthesis.
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An ABI DNA synthesizer equipped with the phosphoramidite monomers was used to synthesized
the oligonuclotide 5ʹ-GTG CG*T GTT TGT-3ʹ, where G* represents the N-(dG-8-yl)-6-AC adduct. The
coupling efficiency of the modified dG was more than 95%. The oligonuclotide was synthesized in DMT-
off mode, where the final 5ʹ-DMT group was cleaved before it was released from solid support. The
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oligomer was then deprotected using standard conditions (overnight at 55 C with NH4OH) except that
0.25M 2-mercaptoethanol was added to avoid oxidative degradation, and the reaction mixture was then
lypholilized. This crude oligonucleotide was purified in reverse-phase HPLC using C-18 semiprep column
(Phenomenex Gemini-NX-5µ) with an eluent of 0.1 M NH4OAc/ 1% CH3CN-H2O (Solvent A) and 80 %
CH3CN/20% H2O (solvent B) with a linear gradient (0 to 40 minutes, 2% to 40 % ) at a flow rate of 4
mL/min monitored at both 254 nm and 350 nm. Purified oligomers were collected in several 1.5 mL
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microcentrifuge tubes, filtered and washed through amicon 3K ultracel filters to remove the excess salt.
Desalted fractions were dried using a speedvac at 40 ° C and characterized with HRMS in negative mode;
12 mer: 5ʹ-GTG CG*T GTT TGT-3ʹ HRMS calcd: 3939.7, found 3939.0 (Figure S2.8 in SI).
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Construction of a plasmid containing N-(dG-8-yl)-6-AC, its replication in HEK 293T cells, and
analyses of progeny. The adduct containing 12-mer 5ʹ-GTG CG*T GTT TGT-3ʹ was incorporated by
enzymatic ligation into a single-stranded pMS2 plasmid by an approach reported earlier.21,24 The DNA
sequence of the dodecamer was chosen from TP53 gene codon 272-275, since the guanine at codon 273
(CG*T) is one of the hot spots of single-base subtitutions in human cancers.25,26 A control plasmid with no
lesion was prepared similarly. The adduct containing or unmodified construct was mixed with an equal
amount of unmodified plasmid DNA, the latter containing a different DNA sequence of the ligated
dodecamer and used as an internal control. The mixed DNA was transfected into HEK 293T cells.
Following incubation of the cells for 24 h to allow for one round of replication of the plasmid, the DNA
was isolated and used to transform E. coli DH10B cells. The percentage of the colonies derived from the
lesion containing plasmid relative to the internal control provided the percentage of TLS, which was
determined by oligonucleotide hybridization followed by DNA sequencing.21,24
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Figure 1. TLS efficiencies of N-(dG-8-yl)-6-AC construct in HEK 293T cells and various TLS polymerase
knockdown HEK 293T cells. The percent TLS in various polymerase knockdown was measured relative to
an internal control in which a different 12-mer was inserted. HEK 293T cells were treated with negative
control (NC) siRNA or siRNA for the single, double, or triple polymerase(s) knockdowns as indicated. The
data represent the means and standard errors of the mean from 2-3 independent replication experiments.
**P < 0.01; ***P < 0.001. The P-values were calculated by using unpaired two-tailed Student’s t-test.
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Roles of TLS polymerases in N-(dG-8-yl)-6-AC bypass. In HEK 293T cells, the TLS efficiency for the
N-(dG-8-yl)-6-AC adduct containing construct was 77±2% compared to the number of progeny generated
from the undamaged construct set at 100% (Figure 1). In hPol η-, hPol κ-, hPol ζ-, and hREV1-deficient
cells, the TLS efficiency was reduced to 66±2%, 45±3%, 37±3%, and 32±2%, respectively (Figure 1),
whereas the TLS efficiency remained statistically unalterd in hPol ι-deficient cells. We also performed the
replication in cells with simultaneous knockdown of two or three TLS polymerases. For simultaneous
deficiency of hPol ζ and hREV1, the TLS efficiency was reduced to 26±3%, whereas simultaneous
deficiency of hPol κ, hPol ζ and hREV1 showed the lowest TLS efficiency of 18±2%, nearly 80% reduction
of TLS compared to that from unaltered HEK 293T cells. This suggests that bypass of N-(dG-8-yl)-6-AC
adduct is primarily carried out by the TLS polymerases, although involvement of other DNA polymerases
cannot be ruled out.
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Figure 2. Mutational frequencies of N-(dG-8-yl)-6-AC construct in HEK 293T cells and various TLS
polymerase knockdown HEK 293T cells. HEK 293T cells were treated with negative control (NC) siRNA
or siRNA for the single, double, or triple polymerase(s) knockdowns as indicated. The data represent the
means and standard errors of the mean from 2-3 independent replication experiments. *P < 0.05; **P <
0.01. The P-values were calculated by using unpaired two-tailed Student’s t-test.
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Error-prone and error-free bypass of N-(dG-8-yl)-6-AC in HEK 293T cells. DNA sequence analyses
showed that 17.8±0.7% of the progeny from HEK 293T cells were mutants (Figure 2), which predominantly
involved targeted mutations (17.3%) at the modified G but also included a low frequency (nearly 0.5%) of
semi-targeted mutations. We define semi-targeted mutations as mutations detected near N-(dG-8-yl)-6-AC,
even when dC was incorporated correctly opposite the lesion. A major part (17.0%) of the targeted
mutations was G→T with a small fraction (0.5%) of G→A (Figure 3), whereas semi-targeted mutations
involved only C→A at the adjacent 5ʹ-C. The semi-targeted C→A appears to be a characteristic of N-(dG-8-
yl)-6-AC, since N-(deoxyguanosin-8-yl)-1-aminopyrene, another C8-dG adduct formed by the NO2-PAH
1-nitropyrene, does not induce this specific semi-targeted mutation at its adjacent 5’-C.24 In most cases, the
semi-targeted mutations are difficult to rationalize, since they are likely induced as a result of the local
DNA helix distortions, which is expected to be unique for each DNA adduct. MF remained unaffected in
hPol η- and hPol ι-deficient cells (Figure 2). In contrast, MF increased to 23.1±1.5% and 32.2±1.2% in hPol
κ- and hREV1-deficient cells, whereas it decreaed to 11.8±1.1% in hPol ζ-deficient cells (Figure 2). This
suggests that hPol κ and hREV1 are involved in error-free TLS of this lesion, whereas hPol ζ performs
error-prone TLS. Although G→T was the dominant targeted mutation in all cases, in hPol ζ- and hREV1-
deficient cells G→A occurred in substantial frequency and constituted a quarter to a third of all mutations
(Figure 3). The progeny from two or three polymerases knockdown cells further confirmed the pattern as
shown in Figure 2. The largest increase in MF to 34.7±0.8% was noted in cells deficient in both hPol κ and
REV1. In contrast, the greatest decrease in MF to 3.7±0.6% was observed in cells with deficiency in hPol
κ, hPol ζ, and REV1. G→T was the predomoinant mutations in all cases (Figure 3). Low level of
semitargeted mutations involved C→A at the adjacent 5ʹ-C [NC (0.5%), hPol κ- (0.4%), hPol ζ/hPol η-
(0.8%) deficient cells] or T→A at the adjacent 3ʹ-T [hPol ζ- (1.2%), hREV1- (2.5%), hPol κ/hPol ζ- (0.9%),
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hPol ζ/hREV1- (0.3%) deficient cells], whereas both types were detected in hPol κ/hREV1- [1.9% C→A
and 1.9% T→A] and hPol κ/hPol ζ/hREV1- [0.3% C→A and 0.3% T→A] deficient cells.
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Figure 3. The types and frequencies of mutations induced by N-(dG-8-yl)-6-AC in HEK 293T cells and
various TLS polymerase knockdown HEK 293T cells. HEK 293T cells were treated with negative control
(NC) siRNA or siRNA for the single, double, or triple polymerase(s) knockdowns as indicated. The data
represent the means and standard errors of the mean from 2-3 independent replication experiments. *P <
0.05; **P < 0.01. The P-values were calculated by using unpaired two-tailed Student’s t-test.
DISCUSSION
In spite of 6-NC’s potent carcinogenicity in experimental animals and strong mutagenicity in bacteria, its
replication in mammalian cells and structural properties have not been investigated. To aid in such studies,
we sought to develop a total synthesis procedure to incorporate N-(dG-8-yl)-6-AC site-specifically in a
desired sequence in DNA. Buchwald-Hartwig coupling yield of 56% was achieved with Pd2(dba)3, BINAP,
and NaOtBu to obtain protected N-(dG-8-yl)-6-AC, but we found the coupled product unstable, which
necessitated special handling. Even so, >80% yield in each of the successive steps was achieved, except in
the last step to synthesize the phosphoramidite monomer of N-(dG-8-yl)-6-AC, which was isolated in 68%
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yield. The protected N-(dG-8-yl)-6-AC was used to synthesize 5ʹ-GTG CG*T GTT TGT-3ʹ, containing the
TP53 gene codon 272-275, which was incorporated in the pMS2 plasmid and replicated in HEK 293T cells.
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Specialized DNA polymerases of the Y-family along with the B-family enzyme pol ζ have been
shown to replicate bulky DNA damages.27-30 The Y-family polymerases adopt the same right-handed
topology as the replicative polymerases (e.g., pol δ and pol ε), and, similar to replicative polymerases, three
carboxylate residues necessary for catalysis exist in the ‘palm’ domain. But they are located in a much
larger active site pocket that can accommodate bulky or distorted DNA lesions on the DNA template,
allowing them to carry out TLS. In addition, they lack proofreading exonuclease activity. As was noted in
the case of other bulky DNA adducts,21,31-33 TLS efficiency for the N-(dG-8-yl)-6-AC construct was
decreased upon knockdown of each bypass polymerase, but substantial reduction was observed in hPol κ-,
hPol ζ-, and hREV1-deficient cells (Figure 1). Therefore, it is not surprising that the most pronounced effect
of decrease in TLS efficiency by more than 75%, i.e., from 77% (in NC cells) to 18%, occurred upon
concurrent knockdown of these three TLS polymerases. This suggests that the major fraction of bypass of
N-(dG-8-yl)-6-AC is carried out by these polymerases.
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The increase in MF upon knockdown of either hPol κ or hREV1 suggests that these two
polymerases bypass the lesion in an error-free manner, which was further substantiated by an additional
increase in MF upon simultaneous knockdown of both polymerases (Figure 2). That REV1 is involved in
error-free bypass is not surprising, since it has been reported to incorporate dCMP opposite dG and abasic
sites.34-37 It promotes predominantly error-free TLS opposite UV lesions in human cells.38 It can act as a
template-independent dC transferase by a mechanism that allows error-free bypass of the bulky dG
adducts.39 REV1 also acts as a scaffold protein that interacts with the TLS polymerases hPol η, κ, and ι.40
The results presented here do not exclude the possibility that hREV1 plays a dual role of dC transferase
opposite N-(dG-8-yl)-6-AC and as a scaffold for the other TLS polymerases.
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In contrast, MF was reduced from 17.5% to 11.6%, a 33% reduction in MF, upon knockdown of
hPol ζ, suggesting its role in error-prone bypass of N-(dG-8-yl)-6-AC. Further decrease in MF by 80%, i.e.,
from 17.5% (in NC cells) to 3.6%, was observed when hPol ζ was knocked down concurrently with hPol κ
& hREV1, which confirmed its dominant role in mutagenesis of this lesion (Figure 2). Previous studies
showed that Pol ζ, which is made up of the catalytic subunit Rev3 and the accessory subunit Rev7 that
stimulates the activity of Rev3, extends efficiently from the correct as well as the incorrect nucleotides
opposite many DNA lesions.41-42 In the case of N-(dG-8-yl)-6-AC, however, based on the known activity
of hPol ζ in TLS, we postulate that hPol ζ preferentially extended the incorrect base dA or dT opposite it
incorporated by another polymerase (Figure 3). However, these results do not rule out the possibility that
hPol ζ carried out both incorporation of the incorrect base and its extension. Although G→T was the
dominant mutation in all types of TLS polymerase-deficient cells, in some cases G→A occurred in
substantial frequency (as in the case of hPol ζ- and hREV1-deficient cells) (Figure 3).
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It is interesting to note that for C8-dG-3-aminobenzanthrone, the bulky C8-dG adduct formed by
3-nitrobenzanthrone, another NO2-PAH compound, hPol η and hPol κ carry out mutagenic TLS, whereas
hPol ζ performs error-free bypass.21 Unlike the current study, MF of C8-dG-3-aminobenzanthrone was
significantly reduced upon knockdown of hREV1, suggesting that hREV1 likely plays a non-catalytic role
by physically interacting with hPol η and hPol κ for the mutagenic bypass. On the other hand, for the C8-
dG adduct formed by 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), it was concluded that hPol κ and
hPol ζ cooperatively carry out the majority of the error-prone TLS, whereas pol η is involved in its error-
free bypass.31 Bypass of the dG-N2 adducts have been shown to have a predictable pattern of error-free and
error-prone TLS in that hPol κ is involved in error-free bypass,43 whereas hPol η, hPol ζ, and hREV1
cooperatively carry out mutagenic TLS.43 In contrast, a pattern for the TLS of C8-dG could not be
determined, and bypass of each lesion at the C8 position of dG thus far showed a unique pattern of how the
TLS polymerases interact with the lesion and incorporate a nucleotide opposite it.44 It is likely that the
structural characteristics of the specific C8-dG adduct play critical roles in its interaction with the TLS
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polymerases in correct and incorrect nucleotide incorporation opposite the lesion. A common feature of
mutagenic bypass of the different C8-dG adducts, however, is that G→T is the major type of mutation, as
was observed in the case of N-(dG-8-yl)-6-AC.
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In conclusion, a total synthesis strategy for the preparation of N-(dG-8-yl)-6-AC-containing
oligonucleotides has been developed. Incorporation of the lesion in a plasmid followed by its replication in
human cells showed that the adduct stalls DNA replication and lesion bypass is primarily dependent on
hPol κ, hPol ζ, and hREV1. Moreover, hPol κ and hREV1 are involved in error-free TLS of this lesion,
whereas hPol ζ performs error-prone bypass.
ASSOCIATED CONTENT
Supporting Information
NMR and MS of the synthesized compounds. This material is available free of charge at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Ashis K. Basu – University of Connecticut, Storrs, Connecticut; orcid.org/0000-0001-7951-4984;
Phone: (860) 486-3965; E-mail: ashis.basu@uconn.edu
Other Authors
Paritosh Pande, Kimberly R. Rebello, Arindom Chatterjee, Spandana Naldiga, University of
Connecticut, Storrs, Connecticut.
Notes
The authors declare no competing financial interest.
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ABBREVIATIONS
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6-NC, 6-nitrochrysene; HEK, human embryonic kidney; NO2-PAHs, Nitropolycyclic aromatic
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hydrocarbons; N-(dG-8-yl)-6-AC, N-(deoxyguanosin-8-yl)-6-aminochrysene; 5-(dG-N2-yl)-6-AC, 5-
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(deoxyguanosin-N2-yl)-6-aminochrysenene;
N-(dA-8-yl)-6-AC,
N-(deoxyadenosin-8-yl)-6-
aminochrysene; NER, nucleotide excision repair; PAH, polynuclear aromatic hydrocarbon; DMT,
dimethoxytrityl; BINAP, rac-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl; DCM, dichloromethane;
PAGE, polyacrylamide gel electrophoresis; NC, negative control; MF, mutation frequency; TLS,
translesion synthesis, IQ, 2-amino-3-methylimidazo[4,5-f]quinolone.
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derived from the nitroreduction of 6-nitrochrysene is more resistant to nucleotide excision repair
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