Syntheses and characterizations of the in vivo replicative bypass and mutagenic properties of the minor-groove O2-alkylthymidine lesions

Article abstract of DOI:10.1093/nar/gku748

Endogenous metabolism, environmental exposure, and treatment with some chemotherapeutic agents can all give rise to DNA alkylation, which can occur on the phosphate backbone as well as the ring nitrogen or exocyclic nitrogen and oxygen atoms of nucleobases. Previous studies showed that the minor-groove O²-alkylated thymidine (O²-alkyldT) lesions are poorly repaired and persist in mammalian tissues. In the present study, we synthesized oligodeoxyribonucleotides harboring seven O²-alkyldT lesions, with the alkyl group being a Me, Et, nPr, iPr, nBu, iBu or sBu, at a defined site and examined the impact of these lesions on DNA replication in Escherichia coli cells. Our results demonstrated that the replication bypass efficiencies of the O²-alkyldT lesions decreased with the chain length of the alkyl group, and these lesions directed promiscuous nucleotide misincorporation in E. coli cells. We also found that deficiency in Pol V, but not Pol II or Pol IV, led to a marked drop in bypass efficiencies for most O²-alkyldT lesions. We further showed that both Pol IV and Pol V were essential for the misincorporation of dCMP opposite these minor-groove DNA lesions, whereas only Pol V was indispensable for the T→A transversion introduced by these lesions. Depletion of Pol II, however, did not lead to any detectable alterations in mutation frequencies for any of the O²-alkyldT lesions. Thus, our study provided important new knowledge about the cytotoxic and mutagenic properties of the O²-alkyldT lesions and revealed the roles of the SOS-induced DNA polymerases in bypassing these lesions in E. coli cells.

Full text of DOI:10.1093/nar/gku748

Published online 12 August 2014
Nucleic Acids Research, 2014, Vol. 42, No. 16 10529–10537
doi: 10.1093/nar/gku748
Syntheses and characterizations of the in vivo
replicative bypass and mutagenic properties of the
minor-groove O2-alkylthymidine lesions
Qianqian Zhai1,†, Pengcheng Wang2,†, Qian Cai2 and Yinsheng Wang1,2,*
1Department of Chemistry, University of California, Riverside, CA 92521, USA and 2Environmental Toxicology
Graduate Program, University of California, Riverside, CA 92521, USA
Received July 10, 2014; Revised July 31, 2014; Accepted August 1, 2014
ABSTRACT
INTRODUCTION
Exposure to a variety of endogenous and exogenous agents
can result in damage to genomic DNA (1). Unrepaired
DNA lesions may block DNA replication and transcription
as well as induce mutations in these processes, which may
contribute to the development of cancer and other human
diseases (1).
Alkylation represents a major type of DNA damage
(2). Both endogenous metabolism and environmental expo-
sure can induce DNA alkylation, and DNA alkylation also
constitutes the major mechanism of action for a number
of commonly prescribed cancer chemotherapeutic agents
(3,4). Hence, understanding how the alkylated DNA lesions
perturb the flow of genetic information is important for re-
vealing the implications of these lesions in the etiology for
the development of human diseases. Such knowledge also
forms the basis for designing better strategies for cancer
treatment.
Depending on the nature of the alkylating agents in-
volved, the size of the alkyl group adducted to nucleobases
varies substantially. For instance, methylation can be in-
duced by a number of anticancer drugs including temozolo-
mide, dacarbazine, streptozotocin and procarbazine, and
chloroethylation can be introduced by anticancer drugs in-
cluding carmustine [bis-(2-chloroethyl)-nitrosourea] and lo-
mustine [(1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea] (5).
On the other hand, bulky pyridyloxobutyl (POB) and
pyridylhydroxybutyl (PHB) groups can be conjugated with
the O2 position of cytosine and thymine as well as the
N7 and O6 positions of guanine by metabolites of to-
bacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-
butanone (NNK) and N’-nitrosonornicotine (NNN) (6–9).
The POB and PHB adducts formed on the O6 of dG and O2
of dT are stable, and they have been detected in the esopha-
gus, lung and liver tissues of rats treated with NNK, NNN,
and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, which
is the major metabolite of NNK (10–12). In this context,
it is worth noting that the size of the alkyl group also affects
Endogenous metabolism, environmental exposure,
and treatment with some chemotherapeutic agents
can all give rise to DNA alkylation, which can oc-
cur on the phosphate backbone as well as the
ring nitrogen or exocyclic nitrogen and oxygen
atoms of nucleobases. Previous studies showed
that the minor-groove O2-alkylated thymidine (O2-
alkyldT) lesions are poorly repaired and persist in
mammalian tissues. In the present study, we syn-
thesized oligodeoxyribonucleotides harboring seven
O2-alkyldT lesions, with the alkyl group being a Me,
Et, nPr, iPr, nBu, iBu or sBu, at a defined site and
examined the impact of these lesions on DNA repli-
cation in Escherichia coli cells. Our results demon-
strated that the replication bypass efficiencies of the
O2-alkyldT lesions decreased with the chain length of
the alkyl group, and these lesions directed promiscu-
ous nucleotide misincorporation in E. coli cells. We
also found that deficiency in Pol V, but not Pol II or
Pol IV, led to a marked drop in bypass efficiencies
for most O2-alkyldT lesions. We further showed that
both Pol IV and Pol V were essential for the misincor-
poration of dCMP opposite these minor-groove DNA
lesions, whereas only Pol V was indispensable for
the T→A transversion introduced by these lesions.
Depletion of Pol II, however, did not lead to any de-
tectable alterations in mutation frequencies for any
of the O2-alkyldT lesions. Thus, our study provided
important new knowledge about the cytotoxic and
mutagenic properties of the O2-alkyldT lesions and
revealed the roles of the SOS-induced DNA poly-
merases in bypassing these lesions in E. coli cells.
*To whom correspondence should be addressed. Tel: +1 951 827 2700; Fax: +1 951 827 4713Email: yinsheng.wang@ucr.edu
†The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
ꢀ
C
The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact
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10530 Nucleic Acids Research, 2014, Vol. 42, No. 16
the distributions of alkylation products formed on different
positions of nucleobases and the phosphodiester backbone
(13).
O
N
N
Multiple lines of evidence suggest that the minor-groove
O2-alkylated thymidine (O2-alkyldT) lesions are poorly re-
paired in mammalian systems. Despite the fact that alkyla-
tion of the O6 position of guanine is more facile than that of
the O4 or O2 position of thymine (14), O2-POBdT and O2-
PHBdT were found to accumulate in lung and liver tissues
of NNK- and NNAL-treated F-344 rats at markedly higher
levels than the corresponding adducts formed at the O6 po-
sition of guanine (11,12), suggesting the poor repair of the
O2-POBdT and O2-PHBdT lesions in mammalian tissues.
The inefficient repair of the minor-groove dT lesions is also
manifested by the observation that O2-EtdT is present at
much higher levels, and persist much longer, than O6-EtdG
in tissues of rats treated with DNA ethylating agents (15–
17). Additionally, O2-EtdT could be detected in human lym-
phocyte DNA, and cigarette smoking led to substantially
elevated levels of this lesion (18).
R
O
dR
2
-CH3
R =
2a
O -MedT (
O2-EtdT (2b)
O2-nPrdT (2c)
)
-CH2CH3
-(CH2)2CH3
-CH(CH3)2
-(CH2)3CH3
2
2d
O -iPrdT (
O2-nBudT (2e)
)
2
-CH(CH3)CH2CH3
-CH2CH(CH3)2
2f
O -sBudT ( )
Some replication studies have been carried out for the O2-
alkyldT lesions. O2-MedT, O2-EtdT and O2-POB-dT were
found to strongly block DNA replication and induce muta-
tions in Escherichia coli cells (19,20). In this vein, our recent
study showed that O2-EtdT differs markedly from its regioi-
someric N3- and O4-EtdT in perturbing the efficiency and
fidelity of DNA replication in E. coli cells, and the different
impacts of three modified nucleosides on DNA replication
are attributed to their unique chemical properties (20). In
the present study, we set out to assess systematically how
the size of the alkyl group incorporated to the minor-groove
O2 position of thymine affects the fidelity and efficiency of
DNA replication, and how replication across these lesions
is modulated by SOS-induced DNA polymerases. Thus, we
incorporated seven O2-alkyldT lesions with varying size of
the alkyl group into single-stranded M13 genome and in-
vestigated how these lesions block DNA replication and in-
duce mutations in Escherichia coli cells. We also assessed
the roles of various SOS-induced DNA polymerases in by-
passing these lesions by conducting the replication studies
in E. coli cells that are deficient in one or more of these poly-
merases.
2
2g
O -iBudT (
)
Scheme 1. The structures of the O2-alkyldT lesions examined in the
present study.
O
O
N
O
N
O
N
N
NH
N
N
R
N
O
R
R
b
c
a
O
O
O
DMTrO
TsO
HO
DMTrO
O
O
O
O
O
P
OH
OH
2a-g
OH
3a-g
(iPr)2
N
O(CH2)2CN
1
4a-g
a
e: R = nBu;
f: R = sec-Bu;
g: R = iBu.
: R = Me;
b: R = Et;
c: R = nPr;
d
: R = iPr;
Scheme 2. Syntheses of phosphoramidite building blocks of O2-
alkylthymidine. Reagents and conditions: (a) ROH, NaHCO3 (with or
without Na), reflux, 16 h; (b) DMTr-Cl, DMAP, pyridine, room tem-
perature, 10 h; (c) 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite,
DIEA, CH2Cl2, 1 h.
and polymerase-deficient AB1157 strains [ꢀpol B1::spec
(Pol II-deficient), ꢀdinB (Pol IV-deficient), ꢀumuC::kan
(Pol V-deficient), and ꢀpol B1::spec ꢀdinB ꢀumuC::kan
(Pol II, Pol IV, Pol V-triple knockout)] were generously pro-
vided by Prof. Graham C. Walker.
MATERIALS AND METHODS
Materials
All chemicals, unless otherwise specified, were from Sigma-
Aldrich (St. Louis, MO) or EMD Millipore. 1,1,1,3,3,3-
Hexafluoro-2-propanol (HFIP) was obtained from TCI
America (Portland, OR). Shrimp alkaline phosphatase and
[␥-32P]ATP were purchased from USB Corporation (Cleve-
land, OH) and Perkin Elmer (Piscataway, NJ), respectively,
and all other enzymes were obtained from New England
Biolabs (Ipswich, MA). All unmodified oligodeoxyribonu-
cleotides (ODNs) were purchased from Integrated DNA
Technologies (Coralville, IA). The 12-mer ODNs harboring
a site-specifically incorporated O2-alkyldT were synthesized
using conventional phosphoramidite chemistry, as detailed
below.
General procedures for syntheses of O2-alkyldT
Phosphoramidite building blocks of seven O2-alkyldT le-
sions (Scheme 1) were synthesized by employing procedures
adapted from those published previously (Scheme 2) (21).
Compound 1 (500 mg, 1.26 mmol), prepared with an iso-
lated yield of 68% according to the published procedures
(22), was suspended in 30 ml of anhydrous alcohol, to
which solution was subsequently added sodium bicarbon-
ate (3.0 eq., 318 mg, 3.78 mmol). For alcohols bearing a
branched alkyl group, i.e. iPrOH, sBuOH and iBuOH, a
catalytic amount of sodium metal (5 mg, 0.217 mmol) was
M13mp7(L2) plasmid and wild-type AB1157 E. coli
strains were kindly provided by Prof. John M. Essigmann,

Nucleic Acids Research, 2014, Vol. 42, No. 16 10531
HPLC
also added. The resulting mixture was refluxed for 16 h
prior to being cooled to room temperature. The solvent was
evaporated under reduced pressure and the residue was pu-
rified by column chromatography with a step gradient of
methanol (0–10%) in CH2Cl2 to afford the desired product.
HPLC separation was performed on an Agilent 1100 system
with a Kinetex XB-C18 column (4.6 × 250 mm, 5 ␮m in par-
˚
ticle size and 100 A in pore size; Phenomenex Inc., Torrance,
CA, USA). For the purification of ODNs, a triethylammo-
nium acetate buffer (50 mM, pH 6.8, solution A) and a
mixture of solution A and acetonitrile (70/30, v/v, solution
B) were employed as mobile phases. The flow rate was 0.8
ml/min, and the gradient profile in terms of solution B was
5–25% in 5 min followed by 25–65% in 60 min. The HPLC
traces for the purifications of the 12-mer lesion-containing
ODNs are shown in Supplementary Figure S19, and elec-
trospray ionization-mass spectrometry (ESI-MS) and tan-
dem MS (MS/MS) characterizations of the purified lesion-
containing ODNs are displayed in Supplementary Figures
S20–S25.
General procedures for dimethoxytrityl (DMTr) protection
of the 5ꢀ-hydroxyl group
Compound 2a-g (100 mg) was dissolved in anhydrous
pyridine (10 ml) and the solution was cooled in an ice
bath. 4-Dimethylaminopyridine (DMAP, 0.5% mol) and
dimethoxytrityl chloride (DMTr-Cl, 1.2 eq.) were subse-
quently added, and the resulting solution was stirred at
room temperature for 10 h. The reaction was quenched with
methanol (0.5 ml), and the solvent removed under reduced
pressure. The residue was purified by silica gel column chro-
matography with ethyl acetate as mobile phase to yield the
desired product 3a-g.
Preparation of the lesion-carrying 22-mer ODNs
The 12-mer O2-alkyldT-containing ODNs were 5ꢁ-
phosphorylated and ligated individually with a 10-mer
ODN (5ꢁ-AGTGGAAGAC-3ꢁ) in the presence of a tem-
plate in the ligation buffer with T4 DNA ligase and ATP at
16◦C for 8 h. The resulting 22-mer ODNs were purified by
denaturing PAGE.
General procedures for phosphoramidite synthesis
To a round bottom flask, which contained a solu-
tion of compound 3a-g (60 mg) in dry CH2Cl2 (3.0
ml) and was stirred in an ice bath, was added N,N-
diisopropylethylamine (DIEA, 2.2 eq.), followed by drop-
wise addition of 2-cyanoethyl-N,N-diisopropyl chlorophos-
phoramidite (1.2 eq.). The mixture was subsequently stirred
at room temperature for 1 h under an argon atmosphere.
The reaction was quenched by cooling the mixture in an
ice bath followed by slow addition of methanol (0.20 ml).
The solution was quickly diluted with ethyl acetate (8.0 ml).
The organic layer was subsequently washed with saturated
NaHCO3 (4.0 ml) and brine (4.0 ml), and dried over anhy-
drous Na2SO4. The solvent was evaporated under reduced
pressure to yield 4a-g in a foam that was directly employed
for ODN synthesis.
Construction of single-stranded lesion-containing and lesion-
free competitor M13 genomes
The lesion-containing and lesion-free M13mp7(L2)
genomes were prepared according to the previously
reported procedures (23). First, 20 pmol of single-
stranded M13 genome was digested with 40 U EcoRI
at 23◦C for 8 h to linearize the vector. The resulting
linearized vector was mixed with two scaffolds, 5ꢁ-
CTTCCACTCACTGAATCATGGTCATAGCTTTC-3ꢁ
and
5ꢁ-AAAACGACGGCCAGTGAATTATAGC-3ꢁ
(25 pmol), each spanning one end of the linearized
vector. To the mixture was subsequently added 30
pmol of the 5ꢁ-phosphorylated 22-mer O2-alkyldT-
bearing ODNs or the competitor ODN (25-mer, 5ꢁ-
AGTGGAAGACATGGCGATAAGCTAT-3ꢁ), and the
DNA was annealed. The resulting mixture was treated with
T4 DNA ligase at 16◦C for 8 h. T4 DNA polymerase (22.5
U) was subsequently added and the mixture was incubated
at 37◦C for 4 h to degrade the excess scaffolds and the
unligated vector. The successfully ligated M13 genomes
were purified from the solution by using Cycle Pure Kit
(Omega). The constructed lesion-containing genomes were
normalized against the lesion-free competitor genome
following published procedures (23).
The reaction yields and spectroscopic characterizations
of the above-synthesized products are provided in the online
Supplementary Materials. The NMR spectra are shown in
Supplementary Figures S1–S18.
ODN synthesis
The
12-mer
lesion-containing
ODNs
5ꢁ-
ATGGCGXGCTAT-3ꢁ (‘X’ represents O2-alkyldT) were
synthesized on a Beckman Oligo 1000S DNA synthesizer
(Fullerton, CA) at 1 ␮mol scale. The above-described phos-
phoramidite building block was dissolved in anhydrous
acetonitrile at a concentration of 0.067 mM. Commercially
available ultramild phosphoramidite building blocks were
used for the incorporation of the unmodified nucleotides
(Glen Research Inc., Sterling, VA, USA) following the
standard ODN assembly protocol. The ODNs were cleaved
from the controlled pore glass support and deprotected
with concentrated ammonium hydroxide at room tem-
perature for 1 h. The solvents were evaporated and the
residues redissolved in water for high-performance liquid
chromatography (HPLC) purification.
Transformation of lesion-containing and competitor M13
genomes into E. coli cells
All the O2-alkyldT-containing M13 genomes, except for the
O2-EtdT-harboring genome which was mixed with the com-
petitor genome at a molar ratio of 20:1, were mixed with
the competitor genome at a molar ratio of 10:1, where a
constant amount (25 fmol) of competitor genome was em-
ployed. The mixtures were transformed into SOS-induced,

10532 Nucleic Acids Research, 2014, Vol. 42, No. 16
region of interest in the ssM13 DNA template. The primers
were 5ꢁ-YCAGCTATGACCATGATTCAGTGAGTGGA-
3ꢁ and 5ꢁ-YTCGGTGCGGGCCTCTTCGCTATTAC-3ꢁ
(Y is an amino group). The amplification cycles were 30,
each cycle consisting of 10 s at 98◦C, 30 s at 65◦C and 15 s
at 72◦C, with a final extension at 72◦C for 5 min. The PCR
products were purified by using Cycle Pure Kit. For the
determination of bypass efficiency, a portion of the above
PCR products was digested with 10 U BbsI restriction en-
donuclease and 1 U shrimp alkaline phosphatase in 10 ␮l
New England Biolabs (NEB) buffer 4 at 37◦C for 30 min,
followed by heating at 80◦C for 20 min to deactivate the
shrimp alkaline phosphatase. To the above mixture was sub-
sequently added a 15 ␮l solution containing 5 mM DTT, 1
␮M ATP (premixed with 1.66 pmol [␥-32P]ATP) and 10 U
T4 polynucleotide kinase, and the mixture was incubated at
37◦C for 30 min, followed by heating at 65◦C for 20 min
to deactivate the T4 polynucleotide kinase. To the resulting
solution was added 10 U MluCI restriction endonuclease,
and the mixture was incubated at 37◦C for 30 min, followed
by quenching with 15 ␮l formamide gel loading buffer con-
taining xylene cyanol FF and bromophenol blue dyes. The
mixture was separated using 30% native polyacrylamide gel
(acrylamide:bis-acrylamide = 19:1), and the DNA bands
were quantified using a Typhoon 9410 variable mode im-
ager.
ATGGCGXGCTAT
Lesion Genome
ATGGCGATAAGCTAT
Competitor Genome
Replication in E. coli cells
ATGGCGXGCTAT
ATGGCGMGCTAT
ATGGCGATAAGCTAT
Progeny of
Lesion Genome
Progeny of
Competitor Genome
PCR amplification
BbsI
MluCI
5’-TTCAGTGAGTGGAAGACATGGCGMGCTATAATTCACTGGCCG-3’
3’-AAGTCACTCACCTTCTGTACCGCNCGATATTAAGTGACCGGC-5’
MluCI
SAP
BbsI
SAP
[γ-
32P]ATP + PNK
[
γ
-
32P]ATP + PNK
BbsI
MluCI
5’-AT p*GGCGMGCTAT AATTCAC-3’
3’-TACCGCp* NCGATATTAA GTG-5’
5’-AT GGCGMGCTAT p*AATTCA-3’
3’-TACCGC NCGATATTAAp* GT-5’
Figure 1. Restriction digestion and post-labeling method for determining
the bypass efficiencies and mutation frequencies of O2-alkyldT lesions in
E. coli cells. ‘X’ in the 22-mer DNA strand designates dT or O2-alkyldT.
The cleavage sites for BbsI and MluCI restriction endonucleases are indi-
cated with broken arrows. Only partial sequence of PCR products for the
lesion-containing genome is displayed, and the PCR products of the com-
petitor genome are not shown. For LC-MS/MS analysis, the PCR prod-
ucts were digested with BbsI and MluCI prior to digestion using shrimp
alkaline phosphatase (SAP), and the [32P]-labeling step involving the use
of T4 polynucleotide kinase (T4 PNK) was omitted.
The above restriction digestion of the PCR prod-
ucts gave rise to the formation of a short duplex
d(p*GGCGMGCTAT)/d(AATTATAGCN),
where
‘M’ represents the nucleobase incorporated at the original
lesion site during DNA replication in vivo, ‘N’ is the
complementary base of ‘M’ in the opposite strand, and
‘p*’ designates the [5ꢁ-32P]-labeled phosphate (Figure 1).
The effect of a lesion on replication fidelity is represented
by the mutation frequencies, i.e. the percentage of mutant
restriction fragments in the total amounts of restriction
fragments released from the PCR products of the lesion-
carrying genome. The bypass efficiency was determined by
using the following formula:
electrocompetent wild-type AB1157 E. coli cells and the iso-
genic E. coli cells that are deficient in Pol II, Pol IV, Pol V
or all three polymerases, following the previously published
procedures (23). In this vein, the SOS system was induced
by irradiating the E. coli cells with 254 nm light at a dose
of 45 J/m2, as described previously (24). We chose to con-
duct the replication experiments in SOS-induced cells be-
cause of the relatively low bypass efficiencies for most of the
O2-alkyldT lesions (vide infra), and we found that the by-
pass efficiency for O2-EtdT in wild-type AB1157 cells was
elevated by ∼3-fold upon SOS induction. The E. coli cells
were subsequently grown in lysogeny broth (LB) culture at
37◦C for 6 h. The phage was recovered from the supernatant
by centrifugation at 13 000 rpm for 5 min and further ampli-
fied in SCS110 E. coli cells to increase the progeny/lesion–
genome ratio. The amplified phage was finally purified us-
ing the QIAprep Spin M13 kit (Qiagen) to obtain the ssM13
DNA template for polymerase chain reaction (PCR) ampli-
fication.
%Bypass Efficiency(%) = (lesion signal/competitor signal)/
(non-lesion control signal/competitor signal) × 100%.
Identification of mutant products by LC-MS/MS
The PCR products were digested with 50 U BbsI restric-
tion endonuclease and 20 U shrimp alkaline phosphatase
in 250 ␮l NEB buffer 4 at 37◦C for 2 h, followed by deacti-
vation of the phosphatase at 80◦C for 20 min. To the mix-
ture was added 50 U MluCI restriction endonuclease, and
the solution was incubated at 37◦C for 1 h. The resulting so-
lution was extracted once with phenol/chloroform/isoamyl
alcohol (25:24:1, v/v). The aqueous layer was subsequently
dried in a Speed-vac, desalted with HPLC and dissolved in
20 ␮l water. A 10 ␮l aliquot was injected for liquid chro-
matography (LC)-MS/MS analysis on an LTQ linear ion
trap mass spectrometer (Thermo Electron, San Jose, CA,
USA). An Agilent Zorbax SB-C18 column (0.5 × 250 mm,
5 ␮m in particle size) was employed, and the gradient for
LC-MS/MS analysis was 5 min of 5–20% methanol fol-
lowed by 35 min of 20–50% methanol in 400 mM HFIP.
Quantification of bypass efficiencies and mutation frequen-
cies
We employed the competitive replication and adduct bypass
(CRAB) assay developed by Delaney et al. (23,25) to assess
the bypass efficiencies of the O2-alkyldT lesions in vivo (Fig-
ure 1). The PCR amplification was carried out with the use
of Phusion high-fidelity DNA polymerase for the sequence

Nucleic Acids Research, 2014, Vol. 42, No. 16 10533
a
The temperature for the ion-transport tube was maintained
at 300◦C. The mass spectrometer was set up for monitor-
ing the fragmentation of the [M-3H]3− ions of the 10-mer
[d(GGCGMGCTAT), where ‘M’ represents A, T, C or G],
and d(AATTATAGCN), with ‘N’ being A, T, C or G. The
fragment ions detected in the MS/MS were assigned man-
ually.
Δ Pol II
WT
T
T
d
u
T
T
T
T
T
d
A
1
T
l
T
d
l
T
T
d
T
T
d
d
d
r
P
d
u
B
T
d
C
G
d
r
o
tr
-
T
C
A
-
o
r
-
d
u
r
d
u
-
-
r
d
u
u
r
r
r
d
t
E
r
r
r
e
e
t
e
t
P
B
B
i
-
e
e
B
P
i
-
e
B
B
n
e
e
m
P
n
i
n
-
2
n
-
2
E
n
M
-
2
m
i
n
s
-
m
M
s
-
2
O
-
2
2
-
2
o
C
-
m
-
-
m
2
2
-
2
o
C
-
2
2
2
m
2
3
0
0
0
O
0
0
O
O
1
1
O
O
O
O
O
O
O
O
1
O
O
1
1
Δ Pol IV
Δ Pol V
RESULTS
T
d
u
T
T
T
d
T
T
d
u
T
d
u
A
1
T
l
l
T
T
T
T
-
d
d
C
G
T
d
T
T
d
t
d
o
A
C
o
d
e
r
P
n
r
-
-
d
r
-
r
P
u
u
B
-
r
e
d
u
d
r
rd
P
r
r
tr
e
r
t
r
t
B
The major objectives of the present study were to explore
how the O2-alkyldT lesions with varying sizes of the alkyl
group compromise DNA replication and to define the roles
of SOS-induced DNA polymerases in bypassing these le-
sions in E. coli cells. To this end, we first synthesized ODNs
harboring site-specifically incorporated O2-alkyldT lesions
(Scheme 1) and characterized these ODNs by ESI-MS and
MS/MS analyses (Supplementary Figures S20–S25). Here,
we use d(ATGGCGXGCTAT) (‘X’ represents O2-nPrdT)
as an example to illustrate how we use ESI-MS and MS/MS
to confirm the site of O2-nPrdT incorporation. Negative-ion
ESI-MS showed that the m/z value for the [M – 3H]3− ion
of the ODN (Supplementary Figure S21a) is 14 Thomson
unit higher than the calculated m/z value of the unmodified
d(ATGGCGTGCTAT), which is consistent with the pres-
ence of an O2-nPrdT in this ODN. The MS/MS of the [M –
3H]3− ion (m/z 1238.5) of the ODN showed the formation
of a series of wn and [an-Base] ions (Supplementary Figure
S21b). The measured molecular masses for the w2, w3, w4
and w5 ions were the same as the calculated ones for the cor-
responding ions of the unmodified ODN, whereas the w6,
w7, w8, w9 and w11 ions exhibited 42 Da higher in molecu-
lar mass than those of the respective fragment ions formed
from the unmodified ODN. These results are in agreement
with the presence of an O2-nPrdT at the 7th position in this
ODN. The above conclusion is further supported by the ob-
served masses for the [an-Base] ions; the measured molecu-
lar masses for the [a8-G], [a9-C] and [a11-A] ions are 42 Da
higher, whereas the measured molecular masses for the [a3-
G], [a4-G], [a5-C], [a6-G] and [a7-X] ions are the same as the
calculated ones for the corresponding fragment ions for the
unmodified d(ATGGCGTGCTAT).
We subsequently ligated these ODNs individually into
single-stranded M13 genome, and assessed the bypass ef-
ficiencies and mutation frequencies of the O2-alkyldT le-
sions by using a modified version of the CRAB and restric-
tion endonuclease and post-labeling (REAP) assays (Fig-
ure 1) (26). In this assay, the progeny genomes emanat-
ing from in vivo replication were amplified by PCR, and
the resulting PCR products were digested with two restric-
tion enzymes, i.e. MluCI and BbsI, and the ODNs re-
leased from the restriction digestion were analyzed by LC-
MS/MS and denaturing PAGE to identify the replication
products, as described recently (20). In this regard, with
the use of 30% native PAGE, we were able to resolve [5ꢁ-
32P]-labeled d(p*GGCGTGCTAT) (non-mutagenic prod-
uct, 10mer-T) from the corresponding products carrying
a T→A or T→G mutation, i.e. d(p*GGCGAGCTAT)
(10mer-A) and d(p*GGCGGGCTAT) (10mer-G) (Figures
1 and 2a and Supplementary Figure S27a). Nevertheless,
the non-mutagenic product co-migrated with the corre-
e
e
e
B
B
s
-
B
e
P
i
-
B
n
i
-
2
e
E
n
i
M
i
n
m
E
n
-
n
-
-
s
m
M
-
2
-
-
2
2
-
-
-
2
o
2
2
2
m
m
2
2
-
-
o
2
3
m
0
2
2
2
m
0
0
0
C
1
0
C
O
O
O
O
O
1
O
O
O
O
O
1
O
O
O
O
1
1
b
ΔPol II
WT
T
T
d
u
T
T
T
T
T
l
T
T
T
d
r
G
l
d
T
d
T
d
u
T
C
0
A
T
d
d
u
d
r
o
T
d
-
-
-
G
d
e
M
r
e
T
o
r
t
n
u
B
-
r
r
u
d
d
u
r
r
-
r
d
t
t
r
rd
r
t
P
e
e
P
B
i
-
e
e
B
B
B
B
P
i
-
e
E
e
P
i
n
n
E
n
m
n
i
-
s
n
s
-
2
-
2
m
M
-
2
O
2
o
-
2
m
m
0
-
2
2
-
2
-
-
-
2
-
2
2
2
-
m
o
2
2
3
m
0
0
C
0
O
O
1
O
C
O
O
1
O
O
1
1
O
O
O
O
O
O
1
1
ΔPol IV
ΔPol V
T
d
T
T
d
u
T
T
T
d
T
d
u
T
l
o
T
G
1
l
T
T
d
A
-
r
T
d
T
T
C
T
d
-
-
T
G
o
r
t
n
rd
P
u
r
r
d
u
d
-
d
u
d
t
u
B
r
r
-
d
rd
P
tr
n
r
r
t
e
e
r
e
e
e
e
P
B
B
B
i
-
B
B
e
P
E
i
e
m
E
i
n
-
n
-
n
i
M
m
n
s
-
-
-
s
-
2
O
2
M
-
2
2
m
m
m
0
1
2
o
-
2
2
-
-
2
2
-
-
2
2
o
-
2
2
m
2
3
0
0
C
0
O
1
0
C
O
O
O
O
O
1
1
O
O
O
O
O
O
O
1
Figure 2. Native PAGE (30%) for monitoring the bypass efficiencies and
mutation frequencies of O2-alkyldT lesions in SOS-induced AB1157 E. coli
cells that are proficient in translesion synthesis or deficient in Pol II, Pol
IV or Pol V. (a) Gel image showing the 13-mer and 10-mer products re-
leased from the top strand (lesion-containing strand) of the PCR products
of the progeny of the competitor genome and the control or lesion-carrying
genome, where 10mer-A, 10mer-C, 10mer-G and 10mer-T represent the
[5ꢁ-32P]-labeled standard ODNs 5ꢁ-GGCGMGCTAT-3ꢁ, with ‘M’ being
A, C, G and T, respectively, and 10mer-CA designates TG→CA tandem
double mutation. (b) Gel image showing the 13-mer and 10-mer products
released from the bottom strand (opposite to lesion-containing strand) of
the PCR products of the progeny of the competitor genome and the control
or lesion-carrying genome, where 10mer-A, 10mer-C, 10mer-G and 10mer-
T represent the [5ꢁ-32P]-labeled standard ODNs 5ꢁ-AATTATAGCN-3ꢁ,
with ‘N’ being A, C, G and T, respectively, and 10mer-TG designates 5ꢁ-
AATTATAGTG-3ꢁ. The lesion-containing genomes were mixed individ-
ually with the competitor genome at molar ratios of 10:1 or 20:1 in the
transfection experiments (see the Materials and Methods section).
sponding product bearing a T→C mutation at the le-
sion site, i.e. d(p*GGCGCGCTAT) (10mer-C, Figure 2a
and Supplementary Figure S27a). To determine the fre-
quency of T→C mutation, we reversed the order of the
two restriction enzyme digestion so that the bottom strand,
namely d(AATTATAGCN), could be selectively radiola-
beled. In doing so, we could completely resolve the prod-
uct emanating from T→C mutation at the lesion site, i.e.
d(AATTATAGCG) (10mer-G), from the non-mutagenic

10534 Nucleic Acids Research, 2014, Vol. 42, No. 16
35
We also determined the mutation frequencies of the
O2-alkyldT lesions in wild-type and polymerase-deficient
AB1157 E. coli strains (Figure 4). Interestingly, the T→G
mutation was totally abolished when the replication experi-
ments were conducted in E. coli cells that are deficient in Pol
IV or Pol V (Figure 4c), underscoring the mutual involve-
ment of Pol IV and Pol V in the mutagenic incorporation of
dCMP opposite the minor-groove O2-alkyldT lesions and
the subsequent extension of the mismatched primer termi-
nus. Additionally, depletion of Pol V resulted in the com-
plete loss of T→A mutation for all O2-alkyldT lesions (Fig-
ure 4b). Thus, Pol V is essential for the misincorporation
of dTMP opposite these lesions. Individual depletion of
any of the SOS-induced DNA polymerases did not lead to
the elimination of the T→C mutation for any of the O2-
alkyldT lesions, whereas simultaneous deletion of all three
SOS-induced DNA polymerases rendered the T→C muta-
tion undetectable for O2-MedT, O2-nBudT and O2-sBudT
(Figure 4a). These results suggest the redundant roles of Pol
II, Pol IV and Pol V in the misincorporation of dGMP op-
posite these three O2-alkyldT lesions. On the other hand,
no marked difference in T→C mutation was observed for
O2-EtdT, O2-nPrdT, O2-iPrdT or O2-iBudT in wild-type or
triple knockout AB1157 cells (Figure 4a), suggesting the in-
volvement of other polymerase(s) (e.g. Pol III) in the gener-
ation of the T→C mutation for these lesions. Aside from
the absence of difference in bypass efficiencies, the muta-
tion frequencies for all the seven O2-alkyldT lesions were
indistinguishable in the wild-type and Pol II-null AB1157
cells (Figure 4), supporting that Pol II is not responsible for
bypassing the minor-groove O2-alkyldT lesions.
W
W
T
T
Δ
P
P
o
o
l
l
I
I
I
I
Δ
P
P
o
o
l
l
I
I
V
V
Δ P
P
o
ol
l
V
V
Δ
Pol II, IV, V
Pol II, IV, V
30
25
20
15
10
5
0
O2-iBudT
i-bu-dT
Me-dT
O2-MedT
Et-dT
npr-dT
ipr-dT
O2-iPrdT
nbu-dT sec-bu-dT
O2-nBudT O2-sBudT
O2-EtdT O2-nPrdT
Figure 3. The bypass efficiencies of the O2-alkyldT lesions in E. coli strains
that are proficient in translesion synthesis or deficient in Pol II, Pol IV,
Pol V or all three SOS-induced DNA polymerases. The data represent the
means and standard deviations of results from at least three independent
replication experiments.
product (10mer-A) and the products with a T→A (10mer-
T) or T→G (10mer-C) mutation (Figure 2b and Supple-
mentary Figure S27b). Thus, by monitoring separately the
products from the two strands, we were able to quantify the
frequencies of nucleobase substitutions.
We further confirmed the identities of the above restric-
tion digestion products by using LC-MS/MS, where we
monitored the fragmentation of the [M – 3H]3− ions of
d(GGCGMGCTAT) and d(AATTATAGCN), where ‘M’
and ‘N’ designate the nucleotides inserted at the initial dam-
age site and the opposing nucleotide in the complementary
strand, respectively. Our LC-MS/MS data revealed that,
similar as what we found recently for the O2-EtdT (20), all
other six O2-alkyldT lesions display promiscuous miscod-
ing properties, where T→A, T→C and T→G mutations
were observed (representative LC-MS and MS/MS data are
shown in Supplementary Figures S28–S30). Additionally,
O2-iPrdT induced a unique type of mutation, the TG→CA
tandem mutation (Supplementary Figures S28–S30).
After identifying the replication products, we determined
the bypass efficiencies of the O2-alkyldT lesions by mea-
suring the ratio of the total amounts of products from the
lesion-containing genome over the damage-free competitor
genome and normalized this ratio against the correspond-
ing ratio obtained for the damage-free control genome, as
described in the Materials and Methods section. Our re-
sults showed that, in SOS-induced wild-type E. coli cells, the
bypass efficiency decreases with the size of the alkyl group
(Figure 3).
DISCUSSION
We assessed systematically the cytotoxic and mutagenic
properties of the O2-alkyldT lesions and our results led to
several important conclusions. First, we found that the O2-
alkyldT lesions are strong impediments to DNA replication
in E. coli cells, with the blocking effect increasing with the
size of the alkyl group. This may reflect the greater diffi-
culty experienced by the DNA polymerases in accommo-
dating the more bulky O2-alkyldT lesions into their active
sites, which results in less productive nucleotide incorpora-
tion at or near the lesion site.
Second, our results demonstrated that the nucleotide in-
sertions opposite the O2-alkyldT lesions are promiscuous,
where all three types of single-base substitutions, namely
T→A, T→C and T→G mutations, were observed for
each of the O2-alkyldT lesion. The promiscuous nucleotide
misinsertion opposite the O2-alkyldT lesions is in line with
the notion that the incorporation of an alkyl group to the O2
position of thymine may render the nucleobase unfavorable
in pairing with any of the four normal nucleobases (27).
Third, we observed that the misincorporation of dTMP
opposite these lesions required Pol V, whereas dCMP misin-
sertion necessitates both Pol IV and Pol V. In this respect,
it is worth noting that replication across the (+)-trans-anti
diastereomer of the benzo[a]pyrene-7,8-diol-9,10-epoxide
(BPDE) adduct on minor-groove N2 position of dG neces-
sitates the mutual action of Pol IV and Pol V (28), whereas
only Pol IV is essential for the replicative bypass of the
To define the roles of the SOS-induced DNA polymerases
in bypassing the O2-alkyldT lesions, we conducted the repli-
cation experiments in E. coli strains deficient in various
SOS-induced DNA polymerases. Our results showed that
the removal of Pol II did not affect appreciably the bypass
efficiencies for any of the O2-alkyldT lesions; however, pro-
nounced drops in bypass efficiencies were observed in Pol
V-null cells, supporting that Pol V plays a major role in by-
passing the minor-groove O2-alkyldT lesions (Figure 3).

Nucleic Acids Research, 2014, Vol. 42, No. 16 10535
a
b
80
70
60
50
40
30
20
10
0
30
25
20
15
10
5
W
W
T
T
Δ
P
P
o
o
l
l
IIII
Δ
P
P
o
o
l
l
I
I
V
V
Δ PPo
o
l
l
V
V
Pol IIII, IVIV, V
Δ Pol , , V
W
W
T
T
Δ
P
P
o
o
l
l
IIII
Δ
P
P
o
o
l
l
IVIV
Δ
P
P
o
o
l
l
V
V
Pol II, , V
Δ Pol II,IVIV, V
0
Methyl-dT
Ethyl-dT n-propyl-dT i-propyl-dT n-butyl-dT sec-butyl-dT i-butyl-dT
O2-MedT O2-EtdT O2-nPrdT O2-iPrdT O2-nBudT O2-sBudT O2-iBudT
O2-MedT O2-EtdT O2-nPrdT O2-iPrdT O2-nBudTO2-sBudT O2-iBudT
Methyl-dT
Ethyl-dT n-propyl-dT i-propyl-dT n-butyl-dT sec-butyl-dT i-butyl-dT
c
d
15
10
5
25
20
15
10
5
W
W
T
T
Δ
P
P
o
o
l
l
II
I
Δ
P
P
o
o
l
l
I
I
V
V
Δ
Po
o
l
l
V
V
Δ
Pol II, , V
Pol II, IVIV, V
0
0
WT
WT
¦¤Pol II
Δ pol II
¦¤Pol IV
Δ pol IV
¦¤Pol V
Δ pol V
¦¤Pol II, IV, V
Δ pol II, IV, V
Methyl-dT
Ethyl-dT
n-propyl-dT i-propyl-dT n-butyl-dT sec-butyl-dT i-butyl-dT
O2-MedT O2-EtdT O2-nPrdT O2-iPrdT O2-nBudT O2-sBudT O2-iBudT
Figure 4. Mutation frequencies of the O2-alkyldT lesions in E. coli cells. Shown are the frequencies for the T→C (a), T→A (b) and T→G (c) mutations
observed for the O2-alkyldT lesions, and the TG→CA mutation found for O2-iPrdT (d). The data represent the means and standard deviations of results
from three independent replication experiments.
corresponding (−)trans-anti diastereomer (28–30). Future
structural studies on complexes between Pol IV/Pol V and
lesion-containing primer templates will provide important
mechanistic insights into the unique mutagenic nucleotide
incorporations mediated by Pol V alone, or by the combined
action of Pol IV and Pol V.
It is also worth noting the difference in replicative
bypass of the O2-alkyldT lesions with the more exten-
sively studied minor-groove N2-alkyldG lesions. In this
vein, a site-specifically incorporated N2-(1-carboxyethyl)-
2ꢁ-deoxyguanosine was found not to block strongly the
DNA replication in wild-type AB1157 cells, and depletion
of Pol IV, but not Pol II or Pol V, led to a marked drop
in bypass efficiency for this lesion (26). In addition, bio-
chemical experiments revealed that Pol IV and its human
ortholog, i.e. DNA polymerase ␬, are capable of inserting
the correct nucleotide (dCMP) opposite a number of N2-
dG lesions at high efficiency (26,31–34). This is in keeping
with the findings made from the X-ray crystal structure of
human DNA polymerase ␬ in complex with the primer–
template, which reveals the capacity of the active site of the
polymerase in accommodating the minor-groove modifica-
tions at the primer–template junction (35). Pol IV may be
capable of fitting the O2-alkyldT lesions into its active site.
Different from alkylation on the N2 position of dG, which
does not disrupt significantly the hydrogen bonding proper-
ties of the guanine base, the inability of the O2-alkyldT le-
sions to form favorable base pair with any of the four canon-
ical nucleotides may impede productive nucleotide incorpo-
ration opposite the lesion site. This may account for the lack
of significant involvement of Pol IV in bypassing the minor-
groove dT lesions.
Taken together, our systematic shuttle vector-based study
on a group of structurally defined O2-alkyldT lesions pro-
vided important new knowledge about the impact of this
under-investigated group of DNA lesions on the efficiency
and accuracy of DNA replication in vivo. Our results also
demonstrated that the cytotoxic properties of these DNA
lesions depend on the size of the alkyl group and the muta-
genic properties of these lesions are modulated by the nature
of SOS-induced DNA polymerases involved.

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ACKNOWLEDGMENTS
The authors would like to thank Prof. Graham C. Walker
for providing the E. coli strains and Prof. John M. Essig-
mann for providing the initial M13 vector.
FUNDING
National Institutes of Health (NIH) [ES025121 to Y.W.].
Funding for open access charge: NIH [R01 ES025121].
Conflict of interest statement. None declared.
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