Cytotoxic and mutagenic properties of O4-alkylthymidine lesions in Escherichia colicells

Article abstract of DOI:10.1093/nar/gkv941

Due to the abundant presence of alkylating agents in living cells and the environment, DNA alkylation is generally unavoidable. Among the alkylated DNA lesions, O⁴-alkylthymidine (O⁴-alkyldT) are known to be highly mutagenic and persistent in mammalian tissues. Not much is known about how the structures of the alkyl group affect the repair and replicative bypass of the O⁴-alkyldT lesions, or how the latter process is modulated by translesion synthesis polymerases. Herein, we synthesized oligodeoxyribonucleotides harboring eight site-specifically inserted O⁴-alkyldT lesions and examined their impact on DNA replication in Escherichia colicells. We showed that the replication past all the O⁴-alkyldT lesions except (S)- and (R)-sBudT was highly efficient, and these lesions directed very high frequencies of dGMP misincorporation in E. coli cells. While SOS-induced DNA polymerases play redundant roles in bypassing most of theO⁴-alkyldT lesions, the bypass of (S)- and (R)-sBudT necessitated Pol V. Moreover, Ada was not involved in the repair of any O⁴-alkyldT lesions, Ogt was able to repair O⁴-MedT and, to a lesser extent, O⁴-EtdT and O⁴-nPrdT, but not other O⁴-alkyldT lesions. Together, our study provided important new knowledge about the repair of the O⁴-alkyldT lesions and their recognition by the E. coli replication machinery.

Full text of DOI:10.1093/nar/gkv941

Published online 22 September 2015
Nucleic Acids Research, 2015, Vol. 43, No. 22 10795–10803
doi: 10.1093/nar/gkv941
Cytotoxic and mutagenic properties of
O4-alkylthymidine lesions in Escherichia coli cells
Pengcheng Wang1, Nicholas J. Amato2, Qianqian Zhai2 and Yinsheng Wang1,2,*
1Environmental Toxicology Graduate Program,University of California, Riverside, CA 92521-0403, USA and
2Department of Chemistry, University of California, Riverside, CA 92521-0403, USA
Received June 27, 2015; Revised September 6, 2015; Accepted September 8, 2015
ABSTRACT
groups adducted to DNA varies from a simple methyl group
to complex alkyl functionalities (6,7). Despite the cytotoxic,
teratogenic and carcinogenic effects, alkylating agents also
constitute a major class of cancer chemotherapeutic drugs
(8).
Due to the abundant presence of alkylating agents
in living cells and the environment, DNA alkylation
is generally unavoidable. Among the alkylated DNA
lesions, O4-alkylthymidine (O4-alkyldT) are known to
be highly mutagenic and persistent in mammalian
tissues. Not much is known about how the struc-
tures of the alkyl group affect the repair and replica-
tive bypass of the O4-alkyldT lesions, or how the lat-
ter process is modulated by translesion synthesis
polymerases. Herein, we synthesized oligodeoxyri-
bonucleotides harboring eight site-specifically in-
serted O4-alkyldT lesions and examined their im-
pact on DNA replication in Escherichia coli cells. We
showed that the replication past all the O4-alkyldT le-
sions except (S)- and (R)-sBudT was highly efficient,
and these lesions directed very high frequencies of
dGMP misincorporation in E. coli cells. While SOS-
induced DNA polymerases play redundant roles in
bypassing most of the O4-alkyldT lesions, the bypass
of (S)- and (R)-sBudT necessitated Pol V. Moreover,
Ada was not involved in the repair of any O4-alkyldT
lesions, Ogt was able to repair O4-MedT and, to a
lesser extent, O4-EtdT and O4-nPrdT, but not other
O4-alkyldT lesions. Together, our study provided im-
portant new knowledge about the repair of the O4-
alkyldT lesions and their recognition by the E. coli
replication machinery.
Among all DNA alkylation adducts, O6-alkyl-2ꢀ-
deoxyguanosine (O6-alkyldG) and O4-alkylthymidine
(O4-alkyldT) are known to be highly mutagenic (6,9–11).
Although O6-alkyldG is induced more efficiently than
O4-alkyldT (6) and can be detected both in vivo and in
vitro (12), O4-alkyldT was found to accumulate at higher
levels than O6-alkyldG in cellular and tissue DNA (13–16),
suggesting that O4-alkyldT may be a more or equally
important DNA alkylation adduct. Thus, it is important
to understand how the O4-alkyldT lesions perturb the
efficiency and accuracy of DNA replication, and how they
are repaired.
Several shuttle vector studies with the use of site-
specifically incorporated DNA lesions have been conducted
for assessing how O4-alkyldT lesions compromise DNA
replication in Escherichia coli and mammalian cells. In this
vein, it was observed that O4-methyl- and O4-ethyl-dT (O4-
MedT and O4-EtdT, Scheme 1) could direct high frequen-
cies of dGMP misincorporation in E. coli cells (17–20).
Likewise, O4-MedT, O4-EtdT and O4-n-propryl-dT (O4-
nPrdT, Scheme 1) were found to be highly mutagenic dur-
ing replication in mammalian cells (21–24), with the former
two inducing T→C transition mutations at higher frequen-
cies (∼20%) than O4-nPrdT (∼12%) (23). The relatively low
mutation frequency of O4-nPrdT was attributed to the more
efficient repair of this lesion, presumably by the nucleotide
excision repair pathway (23). The miscoding properties of
these lesions revealed from the in vivo replication experi-
ments are in keeping with the findings made from in vitro
replication studies, where O4-MedT, O4-EtdT and O4-iso-
propyl-dT (O4-iPrdT, Scheme 1) were shown to direct pref-
erential misincorporation of dGMP by purified DNA poly-
merases (25–27). However, very little is known about the
roles of translesion synthesis DNA polymerases in bypass-
ing these lesions in cells (20).
INTRODUCTION
The genomic integrity is constantly challenged by endoge-
nous metabolism and environmental exposure, leading to a
diverse array of damage products in DNA (1,2). Alkylation
of DNA is generally unavoidable owing to the ubiquitous
presence of alkylating agents in the environment and in liv-
ing cells, generating adducts at multiple sites on nucleobases
as well as the phosphate backbone (3–6). Depending on the
nature of the alkylating agents involved, the size of alkyl
Previous studies showed that O6-alkylguanine-DNA
alkyltransferase (AGT) was relatively inefficient in repair-
*To whom correspondence should be addressed. Tel: +1 951 827 2700; Fax: +1 951 827 4713; Email: Yinsheng.Wang@ucr.edu
ꢁ
C
The Author(s) 2015. 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/4.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

10796 Nucleic Acids Research, 2015, Vol. 43, No. 22
DNA replication, and how replication past these lesions
is affected by AGT proteins and SOS-induced DNA poly-
merases.
MATERIALS AND METHODS
Material
All chemicals, unless otherwise specified, were purchased
from Sigma-Aldrich (St Louis, MO, USA) or EMD
Millipore (Billerica, MA, USA). 1,1,1,3,3,3-Hexafluoro-2-
propanol (HFIP) was obtained from Oakwood Products
Inc. (West Columbia, SC, USA). Common reagents
for solid-phase DNA synthesis were obtained from
Glen Research Co. (Sterling, VA, USA) and unmodified
oligodeoxyribonucleotides (ODNs) were from Integrated
DNA Technologies (Coralville, IA, USA). [␥-32P]ATP
was obtained from Perkin Elmer (Piscataway, NJ, USA).
Shrimp alkaline phosphatase was purchased from USB
Corporation (Cleveland, OH, USA) and all other enzymes
were obtained from New England Biolabs (Ipswich, MA,
USA).
M13mp7(L2), wild-type AB1157 and C215 E. coli
strains, and Ada- and Ogt-deficient E. coli strains (as FC215
derivatives), including C216 (ꢀogt::kan), C217 (ꢀada
ꢀalkB::cam) and C218 (ꢀogt::kan ꢀada ꢀalkB::cam),
were kindly provided by Prof. John M. Essigmann (35).
Polymerase-deficient AB1157 strains [ꢀpol B1::spec (Pol II-
deficient), ꢀdinB (Pol IV-deficient), ꢀumuC::kan (Pol V-
deficient) and ꢀpol B1::specΔdinB ꢀumuC::kan(Pol II, Pol
IV, Pol V-triple knockout)] were generously provided by
Prof. Graham C. Walker (36).
Scheme 1. Structures of the O4-alkyldT lesions examined in the present
study.
ing the O4-alkyldT lesions (28–32). In vitro biochemical as-
says revealed that purified E. coli Ada repairs O4-MedT
10 000 times slower than O6-MedG, and Ogt repairs O4-
MedT at a ∼84-fold higher efficiency than Ada (29,30). The
lack of involvement of Ada in repairing O4-MedT was also
manifested by the observation that the mutation frequency
of O4-MedT, when situated in a single-stranded plasmid,
was not altered by the depletion of this repair protein in
E. coli cells (17). In addition, a recent study showed that
the T:A→C:G transition mutations induced by ethylating
agents and propylating agents in E. coli cells could be effi-
ciently reduced by Ogt, suggesting the involvement of Ogt
in repairing O4-EtdT and O4-nPrdT (32). Considering that
these alkylating agents could also induce alkylation at ade-
nine and at other nucleophilic sites (i.e., O2 and N3) on
thymine, it is not clear whether the reduction in T:A→C:G
transition mutations is primarily attributed to the repair of
O4-alkyldT lesions. Thus, it is important to assess system-
atically the AGT-mediated repair of O4-alkyldT lesions and
the modulation of this repair by the structure of the alkyl
group.
Although site-specific mutagenesis studies have been car-
ried out for several O4-alkyldT lesions, the studies were con-
ducted with the use of somewhat disparate shuttle vector
systems relying on colony picking/counting and sequenc-
ing to estimate the degrees to which these lesions impede
DNA replication and induce mutations, respectively. In the
present study, we set out to assess comprehensively, by us-
ing a highly quantitative and accurate shuttle vector-based
assay (33,34), how the size (from methyl to butyl), branch-
ing (iPr, iBu and sBu versus their straight chain counter-
parts) and stereochemsitry (R versus S diastereomers of
sBu) of the alkyl group incorporated to the major-groove
O4 position of thymine affect the fidelity and efficiency of
Chemical syntheses
Eight O4-alkyldT derivatives were synthesized (Scheme 1).
The synthetic route for the phosphoramidite building
blocks of the O4-alkyldT lesions was adapted from previ-
ously published procedures (Scheme 2) (37).
General procedures for the syntheses of O4-alkylthymidine
Compound 1 (300 mg, 0.58 mmol), prepared at an overall
yield of 81% according to the published procedures (37),
was dissolved in 6 ml of anhydrous acetonitrile at 0◦C, to
which solution were added 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU, 0.17 ml, 1.16 mmol) and the corresponding alco-
hol (1 ml). After 30 min, the resulting mixture was allowed
to warm to r.t. and the solution was stirred overnight. The
solution was neutralized by addition of 0.5 M aqueous so-
lution of KH2PO4 (pH 6.5) and the product was extracted
into chloroform (40 ml). The organic layer was then washed
with brine (3 × 20 ml) and dried over anhydrous Na2SO4.
The filtrate was concentrated and the residue was dissolved
in 4 ml of anhydrous tetrahydrofuran (THF), to which 0.26
ml of tetrabutylammonium fluoride (TBAF) in THF (1.0
M) was added. The resulting mixture was stirred for another
2 h and concentrated. The residue was purified by silica gel
column chromatography with a step gradient of methanol
(0–7%) in methylene chloride to afford the desired product
2a-h. In this context, the stereospecific syntheses of O4-(S)-
sBudT and O4-(R)-sBudT were accomplished by employing

Nucleic Acids Research, 2015, Vol. 43, No. 22 10797
Scheme 2. Syntheses of phosphoramidite building blocks of O4-alkylthymidine␣. ␣Reagents and conditions: (a) ROH, DBU, r.t., 10 h; (b) TBAF, THF,
r.t., 1 h; (c) DMTr-Cl, DMAP, pyridine, r.t., 10 h; (d) 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite, DIEA, CH2Cl2, 1 h.
the commercially available (S)-sBuOH and (R)-sBuOH, re-
spectively.
synthesized phosphoramidite building block was dissolved
in anhydrous acetonitrile at a concentration of 0.067 M.
Commercially available phosphoramidite building blocks
(ultramild) were employed for the incorporation of the
unmodified nucleotides (Glen Research Co., Sterling, VA,
USA) following the standard ODN assembly protocol. The
ODNs were cleaved from the controlled pore glass (CPG)
support and deprotected with concentrated ammonium
hydroxide at r.t. for 1 h. The solvents were evaporated,
and the residues were redissolved in water and purified by
high-performance liquid chromatography (HPLC).
General procedures for DMTr protection
To 100 mg of compound 2a-h, which was dried three
times by evaporation with pyridine and dissolved in an-
hydrous pyridine (10 ml) on an ice bath, were added
4-dimethylaminopyridine (DMAP, 0.5% mol) and 4,4ꢀ-
dimethoxytrityl chloride (DMTr-Cl, 1.2 eq.). The resulting
solution was stirred at room temperature for 10 h. The re-
action was then quenched with methanol (0.5 ml), and the
solvent was removed in vacuo. The residue was purified by
silica gel column chromatography with ethyl acetate as the
mobile phase to yield the desired product 3a-h.
HPLC
HPLC separations were conducted on an Agilent 1100
HPLC system with a Kinetex XB-C18 column (4.60 × 150
˚
mm, 5 ␮m in particle size and 100 A in pore size; Phe-
General procedures for phosphoramidite synthesis
nomenex Inc., Torrance, CA, USA). For the purification
of ODNs, a triethylammonium acetate buffer (50 mM, pH
6.8, Solution A) and a mixture of solution A and ace-
tonitrile (70/30, v/v, Solution B) were employed as mobile
phases. The flow rate was 0.8 ml/min and the gradient pro-
file was 5–25% B in 5 min followed by 25–55% B in 60
min. The HPLC traces for the purifications of the 12-mer
lesion-containing ODNs are shown in Supplementary Fig-
ure S19 and the electrospray ionization-mass spectrometry
(ESI-MS) and tandem MS (MS/MS) of the purified lesion-
containing ODNs are displayed in Supplementary Figures
S20–S26.
To a round bottom flask, which was stirred in an ice bath
and contained a solution of compounds 3a-h (60 mg) in
anhydrous methylene chloride (3.0 ml), were added N,N-
diisopropylethylamine (DIEA, 2.2 eq.) and 2-cyanoethyl-
N,N-diisopropyl chlorophosphoramidite (1.2 eq.). The
mixture was then stirred at room temperature for 1 h under
an argon atmosphere. The reaction was quenched by cool-
ing 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 washed sequen-
tially with saturated NaHCO3 (4.0 ml) and brine (4.0 ml),
and dried over anhydrous Na2SO4. The solvent was evap-
orated under reduced pressure to yield 4a-h in a foam that
was used directly for ODN synthesis.
Preparation of the lesion-carrying 22-mer ODNs
The reaction yields and spectroscopic characterizations
of the above-synthesized products are provided in the online
Supplementary Materials. The nuclear magnetic resonance
spectra for these compounds are shown in Supplementary
Figures S1–S18.
The 12-mer O4-alkyldT-containing ODNs were 5ꢀ-
phosphorylated and ligated individually with a 10-mer
ODN (5ꢀ-AGTGGAAGAC-3ꢀ) 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.
ODN synthesis
Construction of single-stranded lesion-containing and lesion-
free competitor M13 genomes
The
12-mer
lesion-containing
ODNs
5ꢀ-
ATGGCGXGCTAT-3ꢀ (‘X’ represents the O4-alkyldT
lesions) were synthesized on a Beckman Oligo 1000S
DNA synthesizer (Fullerton, CA) at 1 ␮mol scale. The
The lesion-containing and lesion-free M13mp7(L2)
genomes were prepared following the previously reported

10798 Nucleic Acids Research, 2015, Vol. 43, No. 22
(‘Y’ represents a 5ꢀ-amino modifier, i.e., H2N(CH2)6-,
added to the 5ꢀ phosphate group of the ODNs). The am-
plification cycles were 30, with each cycle consisting of 10 s
at 98◦C, 30 s at 65◦C and 15 s at 72◦C, along with a final ex-
tension at 72◦C for 5 min. The PCR products were purified
by using Cycle Pure Kit (Omega). For the determination
of bypass efficiency, a portion of the above PCR products
was digested with 10 U BbsI restriction endonuclease and 1
U shrimp alkaline phosphatase in 10 ␮l New England Bi-
olabs (NEB) CutSmart buffer at 37◦C for 30 min, followed
by heating at 80◦C for 20 min to deactivate the phosphatase.
To the above mixture were subsequently added 5 mM DTT,
1 ␮M ATP (premixed with 1.66 pmol [␥-32P]ATP) and 10 U
T4 polynucleotide kinase in a 15 ␮l solution 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 restric-
tion endonuclease, and the mixture was incubated at 37◦C
for 30 min, followed by quenching with 15 ␮l formamide
gel loading buffer containing xylene cyanol FF and bro-
mophenol blue dyes. The mixture was separated by 30% na-
tive polyacrylamide gel (acrylamide:bis-acrylamide = 19:1).
The DNA bands were quantified using a Typhoon 9410
variable mode Imager.
procedures (Supplementary Figure S27) (38). 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 result-
ing linearized vector was then mixed with two scaffolds,
5ꢀ-CTTCCACTCACTGAATCATGGTCATAGCTTTC-
3ꢀ and 5ꢀ-AAAACGACGGCCAGTGAATTATAGC-3ꢀ
(25 pmol), each spanning one end of the linearized vec-
tor. To the mixture a 30 pmol of the 5ꢀ-phosphorylated
22-mer O4-alkyldT-bearing ODN or the competitor ODN
(25-mer, 5ꢀ-GCAGGATGTCATGGCGATAAGCTAT-3ꢀ)
was subsequently added, and the DNA was annealed. The
resulting mixture was treated with T4 DNA ligase at 16◦C
for 8 h, followed by incubation with T4 DNA polymerase
(22.5 U) at 37◦C for 4 h to degrade the excess scaffolds
and the unligated vector. The lesion-containing and the
lesion-free 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 (38).
Transfection of control, lesion-containing and competitor
M13 genomes into E. coli cells
The control lesion-free M13 genome was mixed with com-
petitor genome at a molar ratio of 1:1, the M13 genomes
containing the O4-alkyldT lesions with the alkyl group be-
ing Me, Et, nPr and nBu were mixed individually with the
competitor genome at a molar ratio of 2:1, and those with
the alkyl group being iPr, iBu, (S)-sBu and (R)-sBu were
mixed separately with the competitor genome at a mo-
lar ratio of 5:1 (25 fmol each of competitor genome was
used). The mixtures were transfected into SOS-induced,
electrocompetent wild-type AB1157 E. coli cells and the
isogenic E. coli cells that are deficient in Pol II, Pol IV,
Pol V or all three polymerases, as well as wild-type E.
coli cells and isogenic strains that are deficient in Ogt,
Ada or both, following the previously published proce-
dures (38). The SOS induction was achieved by irradiat-
ing the E. coli cells with 254 nm light at a dose of 45
J/m2 (36). The E. coli cells were subsequently grown in
lysogeny broth (LB) medium at 37◦C for 6 h. The phage
was recovered from the supernatant by centrifugation at
13 000 r.p.m. for 5 min and further amplified in SCS110
E. coli cells to increase the progeny/lesion-genome ratio.
The amplified phage was finally purified using the QI-
Aprep Spin M13 kit (Qiagen) to obtain the single-stranded
M13 DNA template for polymerase chain reaction
(PCR) amplification.
The
PCR products gave rise to
d(p*GGCGMGCTAT)/d(AATTATAGCN),
above
restriction
digestion
short duplex,
where
of
the
a
‘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 [5ꢀ-32P]-labeled phosphate (Supplementary
Figure S28). The bypass efficiency was determined by using
the following formula:
Bypass efficiency (%) = (lesion signal/competitor
signal)/(non-lesion control signal/competitor signal) ×
100%.
Identification of mutagenic products by LC-MS/MS
The PCR products were digested with 50 U each of BbsI
and MluCI, along with 20 U shrimp alkaline phosphatase in
250 ␮l NEB CutSmart buffer at 37◦C for 2 h, followed by de-
activation of enzymes at 80◦C for 20 min. 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 LC-MS/MS
analysis and an Agilent Zorbax SB-C18 column (0.5 × 250
mm, 5 ␮m in particle size) was used. The gradient for LC-
MS/MS analysis was 5 min of 5–20% methanol followed by
35 min of 20–50% methanol in 400 mM HFIP (pH was ad-
justed to 7.0 with triethylamine). The temperature for the
ion-transport tube was maintained at 300◦C. The LTQ lin-
ear ion trap mass spectrometer (Thermo Electron, San Jose,
CA, USA) was set up for monitoring the fragmentation of
the [M-3H]3− ions of the 10-mer d(GGCGMGCTAT) and
d(AATTATAGCN), with ‘M’ and ‘N’ being ‘A’, ‘T’, ‘C’ or
‘G’. The fragment ions detected in the MS/MS were man-
ually assigned.
Quantification of bypass efficiencies and mutation frequen-
cies
We employed a modified version of the competitive repli-
cation and adduct bypass (CRAB) assay to assess the
bypass efficiencies and mutation frequencies of the O4-
alkyldT lesions upon replication in E. coli cells (Sup-
plementary Figure S28) (33,34,38). The PCR amplifi-
cation was carried out with the use of Phusion high-
fidelity DNA polymerase for the sequence region of inter-
est in the single-stranded M13 DNA template. The primers
were 5ꢀ-YCAGCTATGACCATGATTCAGTGAGTGGA-
3ꢀ and 5ꢀ-YTCGGTGCGGGCCTCTTCGCTATTAC-3ꢀ

Nucleic Acids Research, 2015, Vol. 43, No. 22 10799
BbsI
MluCI
RESULTS
A
5’-ATGGCGMGCTATAATTCA-3’
3’-TACCGCNCGATATTAAGT-5’
The major objectives of this study were to investigate how
the O4-alkyldT lesions with varying structures of the alkyl
group compromise DNA replication and to define the re-
spective roles of SOS-induced DNA polymerases and O6-
alkylguanine-DNA alkyltransferases in bypassing and re-
pairing these lesions in E. coli cells. To this end, we first syn-
thesized lesion-carrying ODNs with a site-specifically in-
corporated O4-alkyldT (Scheme 1) and characterized these
ODNs by ESI-MS and MS/MS analyses (Supplementary
Figures S20–S26). The LC-MS results revealed no de-
tectable level of degradation of O4-alkyldT to dT in these
modified ODNs.
MluCI
BbsI
SAP
SAP
[γ-32P]ATP + PNK
[γ-32P]ATP + PNK
MluCI
BbsI
5’-AT p*GGCGMGCTAT AATTCAC-3’
3’-TACCGCp NCGATATTAA GTG-5’
5’-AT GGCGMGCTAT pAATTCA-3’
3’-TACCGC NCGATATTAAp* GT-5’
B
C
To assess the bypass efficiencies and mutation frequen-
cies of the O4-alkyldT lesions, we ligated the aforemen-
tioned lesion-containing ODNs into single-stranded M13
genome and performed a modified version of the CRAB
and restriction endonuclease and post-labeling (REAP) as-
says to examine how these lesions inhibit DNA replica-
tion and induce mutations in E. coli cells (Supplemen-
tary Figure S28). We employed two restriction enzymes,
i.e. MluCI and BbsI, to digest the PCR products of the
progeny genome, affording 10mer ODN fragment(s) from
the lesion-containing or lesion-free genome and a 13mer
fragment from the competitor genome (Figure 1). The re-
leased ODNs were subjected to LC-MS/MS and native
PAGE analyses to identify the replication products (Supple-
mentary Figures S29–S39), as described elsewhere (20,39).
As illustrated in Figure 1, by switching the order of the
two restriction enzyme digestion, we can selectively incor-
porate a 32P-labeled phosphate to the 5ꢀ ends of lesion-
situated strand, i.e. d(p*GGCGMGCTAT) or its com-
plementary strand, i.e. d(p*AATTATAGCN). When BbsI
was first added, we could resolve, with 30% native PAGE,
the [5ꢀ-32P]-labeled d(p*GGCGTGCTAT) (non-mutagenic
product, 10mer-T) from the products carrying a T→A
or T→G mutation, i.e. d(p*GGCGAGCTAT) (10mer-A)
and d(p*GGCGGGCTAT) (10mer-G). However, the cor-
responding product harboring a T→C mutation at the
lesion site, i.e. d(p*GGCGCGCTAT) (10mer-C), could
not be separated from the non-mutagenic 10mer-T (Fig-
ure 1C and Supplementary Figures S33–S39). On the other
hand, the respective radiolabeled complementary strand,
i.e. d(p*AATTATAGCN), with ‘N’ being ‘A’ or ‘G’, ob-
tained from sequential digestion with MluCI and BbsI,
could be readily resolved from each other (Figure 1B and
Supplementary Figures S33–S39). Thus, with the combina-
tion of the two enzyme digestion procedures, we were able to
distinguish unequivocally the four potential types of repli-
cation products, and by monitoring the products from the
strand of d(p*AATTATAGCN), we could quantify the fre-
quencies of T→C mutation. It is worth noting that our as-
say is also capable of detecting putative −1 or −2 frame-
shift mutation products; such putative frameshift mutations
emanating from the replication of any of the O4-alkyldT-
bearing genomes were, however, below the detection limit
of our method. In this vein, it is of note that large deletions
resulting in the loss of MluCI and/or BbsI recognition site
will not give rise to the restriction fragments used for this
assay, and are thus treated as lack of replicative bypass.
Figure 1. Native PAGE (30%) for monitoring the bypass efficiencies
and mutation frequencies of O4-alkyldT in SOS-induced wild-type (WT)
AB1157 Escherichia coli cells. (A) Sequential restriction enzyme digestion
for the selective labeling of the strand initially bearing the lesion or the
complementary strand. ‘SAP’ and ‘PNK’ designate shrimp alkaline phos-
phatase and T4 polynucleotide kinase, respectively. (B) Gel image showing
the 13-mer and 10-mer products released from the bottom-strand (oppo-
site 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 [5ꢀ-32P]-labeled
standard ODNs 5ꢀ-AATTATAGCN-3ꢀ, with ‘N’ being A, C, G and T, re-
spectively. (C) Gel image showing the 13-mer and 10-mer products released
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 [5ꢀ-
32P]-labeled standard ODNs 5ꢀ-GGCGMGCTAT-3ꢀ, with ‘M’ being A, C,
G and T, respectively.
The identities of the above restriction digestion prod-
ucts were also confirmed by LC-MS/MS analyses. In par-
ticular, we monitored the higher-resolution ‘ultra-zoom
scan’ MS for the [M-3H]3− ions of d(GGCGMGCTAT)
and d(AATTATAGCN) and their MS/MS (Supplemen-
tary Figures S29–S32), where ‘M’ and ‘N’ designate the nu-
cleotides inserted at the initial damage site and the oppos-
ing nucleotide in the complementary strand, respectively.
Unlike the promiscuous nucleotide misinsertions directed
by the minor-groove O2-alkyldT lesions (39), our results re-
vealed that the eight O4-alkyldT lesions exclusively induced
T→C transition mutation, which is consistent with the pre-
viously published results (23). Along this line, our previ-
ous study showed that the sensitivity of our LC-MS- and

10800 Nucleic Acids Research, 2015, Vol. 43, No. 22
MS/MS-based assay can allow for detection of a mutation
frequency that is as low as 0.2% (33).
AlkB in wild-type or Ogt-deficient background, however,
did not result in any appreciable increase in mutation fre-
quencies for any of the O4-alkyldT lesions, suggesting the
lack of involvement of Ada in their repair. The lower mu-
tation frequency of O4-MedT than O4-EtdT and O4-nPrdT
in wild-type cells also reveals that the Ogt-mediated repair
of O4-MedT is more efficient than O4-EtdT and O4-nPrdT.
The bypass efficiencies of the O4-alkyldT lesions were
then calculated from the ratio of the combined intensities
of bands observed for the 10-mer products from the lesion-
containing genome over the intensity of the 13-mer prod-
uct from the competitor genome, with the consideration of
the molar ratio of the lesion over competitor genomes em-
ployed in the initial transfection. The bypass efficiencies for
the lesion-carrying genomes were then normalized against
that for the control lesion-free genome as described in the
‘Materials and Methods’ section. It turned out that, except
for the two diastereomers of O4-sBudT, whose replication
bypass efficiencies are ∼5%, other O4-alkyldT lesions are
not strong impediments to DNA replication in wild-type
AB1157 cells (Figure 2A). In addition, the bypass efficien-
cies for the two diastereomers of O4-sBudT are very similar
in wild-type AB1157 cells (Figure 2A).
DISCUSSION
We systematically investigated the cytotoxic and mutagenic
properties of the O4-alkyldT lesions in E. coli cells and
our results led to several important findings. First, we ob-
served that all O4-alkyldT lesions except for (S)- and (R)-
O4-sBudT do not strongly inhibit DNA replication in E. coli
cells. However, the two diastereomers of O4-sBudT are still
bypassed more efficiently than simple O2-MedT (39), sug-
gesting that the major-groove pyrimidine lesions are much
better tolerated than the minor-groove counterparts by the
E. coli DNA replication machinery.
Second, our results demonstrated that the nucleotide in-
sertion opposite O4-alkyldT lesions is specific, where only
T→C mutation was observed for all the eight O4-alkyldT
lesions and the mutation frequencies of the O4-alkyldT le-
sions were not affected by depletion of Pol II, Pol IV, Pol
V or all three polymerases. The mutation frequencies were
also not perturbed by SOS induction in wild-type AB 1157
cells, indicating that the selective dGMP misinsertion may
be attributed to the distinct chemical properties of the O4-
alkyldT lesions. In this vein, the addition of a methyl group
to O4 position of thymine is known to facilitate its favorable
base pairing with guanine (9,40).
Third, our results revealed that the three SOS-induced
DNA polymerases play somewhat redundant roles in by-
passing the O4-alkyldT lesions with the exception of (S)-
and (R)-O4-sBudT, as reflected by the observation that the
bypass efficiencies of these lesions dropped substantially
only upon depletion of all three polymerases. Efficient by-
pass of O4-sBudT lesions, however, necessitates Pol V. In
this connection, Yuan et al. (41) observed that Pol V also
plays an important role in the bypass of O4-carboxymethyl-
dT in E. coli cells.
Fourth, we found that Ogt, but not Ada, is involved in
repairing O4-alkyldT with small straight-chain alkyl func-
tionality, with O4-MedT being much more efficiently re-
paired than O4-EtdT and O4-nPrdT. The nearly 100% T→C
mutation frequency observed for O4-iPrdT, O4-nBudT, O4-
iBudT or O4-sBudT suggest the lack of repair of these le-
sions in E. coli cells. This observation is in line with the
previous observations that Ada is not involved in repairing
O4-alkyldT and that Ogt is slightly better than Ada in toler-
ating large O6-alkyldG adducts (17,29,30). Likewise, all the
eight O4-alkyldT lesions were completely mutagenic in Ogt-
deficient E. coli cells, suggesting the absence of other DNA
repair mechanisms in the removal of these DNA lesions
when they are situated in single-stranded M13 genome. In
this context, it is important to note that the replication
of these lesions in the single-stranded M13 genome dif-
fers from the replication of these lesions in double-stranded
chromosomal DNA in E. coli cells. In particular, some
DNA repair machinery (e.g. nucleotide excision repair) ne-
We also examined the roles of the SOS-induced DNA
polymerases in bypassing the O4-alkyldT lesions by con-
ducting the replication experiments in E. coli strains defi-
cient in these DNA polymerases. Our results showed that,
similar to what we found previously for O4-EtdT (20), no
significant differences in bypass efficiency were observed for
all O4-alkyldT lesions when any of the three SOS-induced
polymerases were individually depleted. However, a marked
reduction in bypass efficiency was observed for the triple
knockout cells (Figure 2A), indicating that Pol II, Pol IV
and Pol V play somewhat redundant roles in bypassing the
O4-alkyldT lesions in E. coli cells.
The results from native PAGE analysis also allowed us
to measure the mutation frequencies of O4-alkyldT lesions
in wild-type and DNA polymerase-deficient AB1157 E. coli
strains (Figure 2B). Our results revealed that all O4-alkyldT
lesions are highly mutagenic, resulting exclusively in T→C
mutation at frequencies of up to nearly 100%. Interestingly,
the mere increase in the size of the alkyl group from methyl
to ethyl led to a two-fold elevation in the T→C mutation fre-
quency (i.e. from ∼40 to 80%). It is also worth noting that
single or even triple depletion of these three polymerases did
not confer any significant change in mutation frequencies,
suggesting that the miscoding property of the O4-alkyldT le-
sions is independent of the nature of polymerases involved.
The above replication experiments showed that mutation
frequencies for the O4-alkyldT lesions with small straight-
chain alkyl groups (i.e., O4-MedT, O4-EtdT and O4-nPrdT)
were not as high as the other lesions investigated. This might
be attributed to the differential repair of the O4-alkyldT
lesions. To test this, we exploited the involvement of O6-
alkylguanine-DNA alkyltransferases in the repair of O4-
MedT, O4-EtdT, O4-nPrdT and O4-nBudT lesions by con-
ducting the replication experiments in isogenic E. coli cells
that are proficient or deficient in Ogt and/or Ada (Fig-
ure 3). We found that the bypass efficiencies for the four O4-
alkyldT lesions were not significantly altered upon the de-
pletion of these DNA repair factors (Figure 3A). However,
the removal of Ogt in the wild-type or Ada/AlkB-double
knockout background gave rise to elevations in T→C mu-
tation frequencies of O4-MedT, O4-EtdT and O4-nPrdT to
nearly 100%, supporting the involvement of Ogt in the re-
pair of these lesions (Figure 3B). Dual depletion of Ada and

Nucleic Acids Research, 2015, Vol. 43, No. 22 10801
Figure 2. The bypass efficiencies (A) and mutation frequencies (B) of the O4-alkyldT lesions in AB1157 Escherichia 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 three independent replication experiments.

10802 Nucleic Acids Research, 2015, Vol. 43, No. 22
a methyl group (as in O4-iPrdT), located at the ␣-carbon of
the O4 of thymine may render it difficult for the modified
thymine to fit into the active sites of DNA polymerases.
In summary, our systematic shuttle vector-based study
of a group of structurally defined O4-alkyldT lesions pro-
vided important new insights into the impact of this group
of DNA lesions on the efficiency and accuracy of DNA
replication and the repair of these lesions in vivo. Our re-
sults also indicated that the carcinogenic potentials of these
lesions may arise, at least in part, from their high bypass effi-
ciencies and strong mutagenic effects. It will be important to
assess how the O4-alkyldT lesions compromise DNA repli-
cation and how they are repaired in mammalian cells in the
future.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENT
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) [R01 ES025121, T32
ES018827]. Funding for open access charge: NIH [R01
ES025121].
Conflict of interest statement. None declared.
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