A cyclobutane thymine-N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis

Article abstract of DOI:10.1093/nar/gkt1039

Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-Alkylation of the exocyclic amino group of 2′-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (～7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.

Full text of DOI:10.1093/nar/gkt1039

Published online 31 October 2013
Nucleic Acids Research, 2014, Vol. 42, No. 3 2075–2084
doi:10.1093/nar/gkt1039
A cyclobutane thymine–N4-methylcytosine dimer
is resistant to hydrolysis but strongly blocks
DNA synthesis
Junpei Yamamoto1, Tomoko Oyama1, Tomohiro Kunishi1, Chikahide Masutani2,
Fumio Hanaoka3 and Shigenori Iwai1,*
1Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama,
2
Toyonaka, Osaka 560-8531, Japan, Research Institute of Environmental Medicine, Nagoya University,
3
Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan and Faculty of Science, Gakushuin University, 1-5-1 Mejiro,
Toshima-ku, Tokyo 171-8588, Japan
Received August 28, 2013; Revised October 9, 2013; Accepted October 10, 2013
cyclobutane pyrimidine dimers (CPDs) and pyrimi-
dine(6–4)pyrimidone photoproducts. The loss of genetic
integrity caused by alterations in the chemical structure
of DNA leads to carcinogenesis and mutagenesis (1). To
protect their genetic information, organisms have de-
veloped UV protection systems. One of these systems is
nucleotide excision repair (NER), in which several
enzymes function together to remove the UV lesions.
The lack of NER activity causes the inherent genetic
disease, xeroderma pigmentosum (XP), which has been
classified into seven complementation groups and a
variant (XPA–G and XPV) (2). Among them, the
protein encoded by XPV is not directly involved in the
NER pathway, but is responsible for translesion DNA
synthesis (TLS), in which special DNA polymerases, the
Y-family polymerases, bypass the lesions during DNA
synthesis instead of the replicative polymerases stalled at
the damaged site (3). Human DNA polymerase Z (hPolZ),
which is the XPV product (4), and its yeast ortholog,
RAD30 (5), are involved in the error-free bypass of the
cis-syn CPD formed at the TT site (T[]T), and incorporate
two adenylates (6). A decade has passed since these
enzymes were found, and now the molecular mechanisms
of TLS are being elucidated (7,8).
The UV-induced DNA lesions are formed at
bipyrimidine sites in DNA. Among the pyrimidine bases,
cytosine plays an important role for functional develop-
ment of organisms by undergoing epigenetic modification
(9). Although cytosine is widely used for gene regulation,
it is the most labile base and undergoes deamination to
irreversibly form uracil, with a reaction rate constant of
1 ꢁ 10ꢂ10 sꢂ1 under physiological conditions (10). Upon
CPD formation, hydrolytic deamination becomes
accelerated to 3.9 ꢁ 10ꢂ5 sꢂ1 (11). The uracil-containing
CPD is easily formed, and can cause the C!T transition
ABSTRACT
Exposure of DNA to ultraviolet light produces
harmful crosslinks between adjacent pyrimidine
bases, to form cyclobutane pyrimidine dimers
(CPDs) and pyrimidine(6–4)pyrimidone photoprod-
ucts. The CPD is frequently formed, and its repair
mechanisms have been exclusively studied by
using a CPD formed at a TT site. On the other
hand, biochemical analyses using CPDs formed
within cytosine-containing sequence contexts are
practically difficult, because saturated cytosine
easily undergoes hydrolytic deamination. Here, we
found that N-alkylation of the exocyclic amino group
of 20-deoxycytidine prevents hydrolysis in CPD for-
mation, and an N-methylated cytosine-containing
CPD was stable enough to be derivatized into its
phosphoramidite building block and incorporated
into oligonucleotides. Kinetic studies of the CPD-
containing oligonucleotide indicated that its
lifetime under physiological conditions is relatively
long (ꢀ7 days). In biochemical analyses using
human DNA polymerase g, incorporation of TMP
opposite the N-methylcytosine moiety of the CPD
was clearly detected, in addition to dGMP incorpor-
ation, and the incorrect TMP incorporation blocked
DNA synthesis. The thermodynamic parameters
confirmed the formation of this unusual base pair.
INTRODUCTION
Ultraviolet (UV) light causes harmful effects in DNA,
such as the formation of UV-induced damage, i.e.
*To whom correspondence should be addressed. Tel: +81 6 6850 6250; Fax: +81 6 6850 6240; Email: iwai@chem.es.osaka-u.ac.jp
ß The Author(s) 2013. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
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2076 Nucleic Acids Research, 2014, Vol. 42, No. 3
Figure 1. Formation of cyclobutane thymine–cytosine dimers modified with (A) acetyl or (B) methyl groups at the exocyclic amino function. In our
previous work (A), the acetyl group clearly accelerated hydrolysis after CPD formation.
mutation (12,13). On the other hand, 5-methylcytosine
(mC) is more stable to hydrolysis than unmodified
cytosine (14,15). Taylor and coworkers addressed the
problem of the lability of the cytosine-containing CPD
by using mC, instead of intact cytosine. They successfully
prepared and purified short oligonucleotides containing
the mC-CPD, and analyzed TLS by yeast and human
DNA polymerase Z (16,17).
Taking into account the fact that the methylation at the
C5 position of cytosine stabilizes the C–N bond, methyla-
tion at the N4 position of cytosine would also reduce the
frequency of hydrolysis, due to the electron-donating
ability of the methyl group. We recently reported that
the modification of the exocyclic amino group with an
electron-withdrawing acetyl group drastically facilitated
the hydrolytic removal of the acetylamino group, even in
the presence of the methyl group at the C5 position
(Figure 1) (18). A thymine-containing CPD was formed,
in this case. Since the modification of this amino group
can directly modulate the electron density of the C4
Scheme 1. Synthesis of the CPD, formed between thymidine and 20-
position, where the nucleophilic attack of a water
deoxy-N4,5-dimethylcytidine.
Reagents
and
conditions:
(a)
molecule occurs, the attachment of an electron-donating
methyl group to the amino group was expected to reduce
the deamination rate. In fact, the stability of N4-
methylcytosine to hydrolysis was suggested in several lit-
eratures (19,20), but no obvious evidence has been
reported thus far.
methylamine/acetonitrile, water, (b) 80% acetic acid, (c) 50-O-DMT-
thymidine-30-methyl-N,N-diisopropylphosphoramidite, tetrazole/aceto-
nitrile, (d) I2, H2O/THF (e) 80% acetic acid and (f) UV (>280 nm),
acetophenone/H2O, acetonitrile.
Here, we report that the photosensitized cycloaddition
of partially protected thymidylyl-(30!50)-20-deoxy-N4,5-
dimethylcytidine (compound 4 in Scheme 1) afforded a
stable CPD, without the loss of the methylamino group
in the cytosine moiety. Furthermore, the obtained CPD,
formed between thymine and N4,5-dimethylcytosine
(T[]mCm), was quite stable under various synthetic
conditions, and thus we successfully synthesized its
phosphoramidite building block and oligonucleotides con-
taining the cis-syn T[]mCm on a DNA synthesizer. These
oligonucleotides were utilized to investigate the thermo-
dynamic and biochemical properties of the cyclobutane
thymine–N4-methylcytosine dimer.
MATERIALS AND METHODS
General
Reagents for DNA synthesis were purchased from
Applied Biosystems Japan (Tokyo, Japan) and Glen
Research (Sterling, VA, USA). All other reagents and
solvents were purchased from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan) or Sigma-Aldrich (St.
Louis, MO, USA). TLC analyses were performed on
Merck Silica gel 60 F254 plates, and the results were
visualized by UV illumination at 254 nm. For column
chromatography, Wakogel C-200 and C-300 were used
for practical and precise separations, respectively. HPLC

Nucleic Acids Research, 2014, Vol. 42, No. 3 2077
analyses were performed on a Shimadzu gradient-type
analytical system equipped with a Shimadzu SPD-
phosphoramidite, were also installed. Phenoxyacetic an-
hydride and 1-methylimidazole were used as the capping
reagents (23). Two types of 12-mers, d(CGTACT[]mCmCA
TGC) (T[]mCm 12-mer) and d(CAT[]mCmAGCACGAC),
and a 30-mer, d(CTCGTCAGCATCT[]mCmCATCATAC
AGTCAGTG) (T[]mCm 30-mer), were synthesized on the
1 mmol scale, by using the standard synthetic program
modified only in the reaction time for the coupling of the
cis-syn T[]mCm building block (20 min). After the synthe-
sis, the supports were treated with a thiophenol solution
(thiophenol/TEA/THF = 1/2/2, v/v/v) for 1 h, successively
washed with THF (1 ml ꢁ 1), methanol (2 ml ꢁ 5) and then
acetonitrile (2 ml ꢁ 3), and treated with 28% ammonia
water (2 ml) at room temperature for 2 h. The obtained
oligonucleotides were analyzed and purified by HPLC
with linear gradients of 5–13% and 7–13% acetonitrile
for the 12-mers and 30-mer, respectively. The column was
run at ambient temperature, to prevent the hydrolysis
of T[]mCm during the purification. The pooled solution
was evaporated to dryness, and the residue was dissolved
in water (500 ml). The solution was passed through
an illustraTM NAP-5 column (GE Healthcare,
Buckinghamshire, UK) to remove TEAA. The T[]mCm
12-mer was characterized by LC-ESI mass spectrometry
(Supplementary Figure S1), and its molecular mass was
determined as m/z 3608.66, which is identical to the
calculated value ([M–H]ꢂ, 3608.40). The two 12-mers con-
taining T[]T, d(CGTACT[]TCATGC) (T[]T 12-mer) and
d(CAT[]TAGCACGAC), were synthesized using the cis-
syn TT-CPD phosphoramidite. A 12-mer oligonucleotide
containing N4,5-dimethylcytosine, d(CATmCmAGCACG
AC), was prepared by using the 4-triazolothymidine
phosphoramidite.
M10AVP photodiode-array detector.
A
Waters
mBondasphere C18 5mm 300 A column (3.9 mm ꢁ150 mm)
was used on this system, at a flow rate of 1.0 ml minꢂ1
with linear gradient of acetonitrile in 0.1 M
˚
,
a
triethylammonium acetate (TEAA) (pH 7.0) generated
over 20 min. 1H and 31P NMR spectra were measured
on a Varian INOVA 500 spectrometer. 1H chemical
shifts were calibrated with tetramethylsilane (TMS) in
the deuterated solvents. In the D2O solution, the
chemical shifts were indirectly calibrated with sodium
4,4-dimethyl-4-silapentane-1-sulfonate, as an external ref-
erence. 31P chemical shifts were calibrated with trimethyl-
phosphate, as an external reference. Two-dimensional
phase-sensitive NOESY and ROESY spectra were
acquired with mixing times of 700 and 350 ms,
respectively. High-resolution mass spectrometry was per-
formed on a JEOL JMS-700 spectrometer with fast atom
bombardment ionization.
Formation of the cis-syn cyclobutane thymine–N 4,
5-dimethylcytosine dimer, T[]mCm
A 1 mM solution of compound 4 dissolved in 50%
aqueous acetonitrile (1.3 l) was prepared, and the
solution was purged with nitrogen for 5 min. After the add-
ition of acetophenone (1.22 ml, 10.4 mmol), the solution
was irradiated in a Pyrex immersion well apparatus
fitted with a 450 -W high-pressure mercury lamp (UM-
452; Ushio, Tokyo, Japan) in a 4ꢃC water bath.
Aliquots (200 ml) of the reaction mixture were sampled
at appropriate intervals and were evaporated to dryness.
The residues were dissolved in water (200 ml), and the
samples were immediately analyzed by HPLC, using a
27.5–36.5% acetonitrile gradient generated over 20 min.
After 2 h, the solution was concentrated to dryness. To
determine the product structures, the crude products
Temperature dependence of the hydrolysis reaction in the
T[]mCm 12-mer
Solutions (1 ml) of the T[]mCm 12-mer (20 nmol), in
180 mM phosphate buffer (pH 7.4), were placed in
plastic tubes and incubated at 50, 80 and 95ꢃC. At appro-
priate intervals, aliquots (50 ml) of the reaction mixture
were immediately analyzed by HPLC. Fractions of the
starting product were plotted on a logarithmic vertical
axis against the incubation time. The Arrhenius plot was
˚
were roughly separated on a Preparative C18 125 A
column (Waters Corporation, Milford, MA, USA)
installed on a BioLogic LP chromatographic system
(Bio-Rad Laboratories, Hercules, CA, USA). An acetoni-
trile gradient of 25–37.5% was generated over 400 min.
Finally, the reaction was repeated six times, using 5.69 g
of compound 4 (8.28 mmol) in total. The collected residues
were purified by column chromatography using Wakogel
C-300 with a stepwise gradient of 0–10% methanol in
CHCl3. The pooled fractions were concentrated to
dryness to give the cis-syn isomer of compound 5. Yield
420 mg (611 mmol, 7.3%).
produced using kobs
.
Translesion synthesis across the cis-syn T[]mCm by human
DNA polymerase g (hPolg)
The 50-32P-labeled primer-template was prepared by
mixing either the 16-mer primer d(CACTGACTGTATG
ATG), the G-ended 17-mer primer (dG17) d(CACTGAC
TGTATGATGG) or the T-ended 17-mer primer (T17)
d(CACTGACTGTATGATGT), labeled at their 50 ends,
with each of the 30-mer templates, d(CTCGTCAGC
ATCXYCATCATACAGTCAGTG), where the under-
lined XY represents the lesion site, at a molar ratio of
1:1. Standard reaction mixtures (10 ml), containing
40 mM Tris–HCl (pH 8.0), 1 mM MgCl2, 100 mM
dNTPs, 10 mM DTT, 0.24 mg mlꢂ1 BSA, 60 mM KCl,
2.5% glycerol, 40 nM 50-32P-labeled primer-template
and His-tagged human DNA polymerase Z (4,6), were
Oligonucleotide synthesis
The phosphoramidite building blocks of the cis-syn
TT-CPD (T[]T) and 4-triazolothymidine were synthesized
according to the previous reports (21,22). These building
blocks and compound 8 (described in Supplementary
Data) were dissolved in anhydrous acetonitrile at a concen-
tration of 0.13 M and were installed on an Applied
Biosystems 3400 DNA synthesizer. Solutions of nucleoside
phosphoramidites for ultramild DNA synthesis (Glen
Research), as well as the base-unprotected thymidine

2078 Nucleic Acids Research, 2014, Vol. 42, No. 3
incubated at 37ꢃC. Mixtures (10 ml) for the reaction of
exonuclease-deficient Klenow fragment (KF), containing
10 mM Tris–HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2,
1 mM dithiothreitol, 40 nM the labeled primer-template
and KF, were incubated at 37ꢃC as well. The reactions
were terminated by the addition of stop solution (10 ml),
containing 95% formamide, 20 mM EDTA, 0.025%
bromophenol blue and 0.025% xylene cyanol. These
mixtures were heated at 95ꢃC for 10 min, and then the
reaction products were separated by electrophoresis on a
20% polyacrylamide/7.5 M urea gel. The dried gels were
analyzed with a GE Healthcare FLA7000 image analyzer.
The kinetic analysis was performed under single-
completed hit conditions (24), essentially according to
the previous report (25). The standard reaction mixtures
(10 ml), containing six different concentrations of dNTPs,
were incubated at 37ꢃC for three different incubation
times, and the reactions were terminated by the addition
of stop solution (10 ml). The reaction products were
analyzed by electrophoresis as mentioned above, and the
amounts of the extended products on the dried gels were
quantified with the Multi Gauge software, version 3.1
(Fuji Film). In the experiment, the concentrations and
the incubation times were appropriately set, so the
amounts of the extended products would not exceed
20% of the total density. The determination of the
velocities at each concentration of dNTPs was performed
three times, and the averaged velocities were plotted
against the concentrations. To obtain the Michaelis par-
ameters, the plot was fitted with the Michaelis–Menten
equation, v = Vmax [S]/(Km+[S]), by the non-linear least
squares approach, calculated with the Origin 7.0 software.
The catalytic rate constants (kcat) were obtained by the
equation kcat = [Vmax (mol of the primer-template)/(mol
of DNA polymerase)].
(Ct/4)+ÁSꢃ/ÁHꢃ was employed for the fitting of the
van’t Hoff plot, on the basis of the bimolecular association
of two non-self-complementary strands (27), and the 1/Tm
values obtained for each Ct were plotted over ln (Ct/4). The
slope (m) and the y-intercept (b) of the linear least-square
fitting correspond to R/ÁHꢃ and ÁSꢃ/ÁHꢃ, respectively.
The Gibbs free energies of the association of the two
strands at 25ꢃC (ÁGꢃ) were obtained by substituting ÁHꢃ
and ÁSꢃ into the equation ÁGꢃ = ÁHꢃ – TDSꢃ. The error
analysis was then performed, and the variances and the
2
covariance of the linear fitting (ꢀm2, ꢀb and ꢀbm, respect-
ively) were first obtained, according to the literature (28).
The variances of ÁHꢃ, ÁSꢃ and ÁGꢃ were calculated
using the previously reported equation (29), and then the
standard deviations were obtained by calculating the
square roots of the variances.
RESULTS
Synthesis of oligonucleotides containing the cis-syn
cyclobutane thymine–N 4,5-dimethylcytosine dimer,
T[]mCm
The thymidylyl-(30!50)-20-deoxy-N4,5-dimethylcytidine
derivative 4, in which the exocyclic amino group of
cytosine was methylated, was synthesized from
a
4-triazolothymidine derivative (Scheme 1), and
photosensitized [2+2] cycloaddition of compound 4 was
performed, according to the previous report (18). We first
tested a different compound possessing a 2-cyanoethyl
group in the internucleoside phosphotriester, as the
starting material for this reaction. However, the HPLC
analysis revealed that the starting material was signifi-
cantly degraded during the UV irradiation (data not
shown), probably due to the loss of the labile 2-cyanoethyl
group upon the irradiation. To improve the stability in the
photoreaction, the 2-cyanoethyl group was replaced by a
methyl group (30).
Thermodynamic analysis of the formation of duplexes
mimicking the primer-template structures
The irradiation of compound 4 with a high-pressure
mercury lamp in the presence of photosensitizer mainly
yielded four peaks in the HPLC analysis after 2 h, and
these products were referred to as peaks i–iv, in order of
elution (Figure 2). The loss of the original absorption
band of compound 4 in the UV absorption spectra of
these products suggested that these products were
cycloadducts (compound 5). To identify their stereochem-
istry, they were roughly purified by reversed-phase column
chromatography. Although these four peaks were not
separated well on the reversed-phase column, we success-
fully isolated the product that yielded peak i. The ROESY
spectrum of the purified product revealed that the charac-
teristic correlations of H10–H6 and H20–H6 in the 50 and 30
components, respectively, as observed in our previous
study (Supplementary Figure S2) (18). This result
showed that the configuration of the cyclobutane ring
between the two bases was trans-syn, with the SYN-
ANTI conformation of the N-glycosidic bonds. Its exact
mass was determined as m/z 688.2770 ([M+H]+; calcd for
C28H47N5O11PSi, 688.2779), which demonstrated that the
methylamino group of the trans-syn (SYN-ANTI)
cycloadduct was intact. The products that yielded peaks
Lesion-containing 12-mers, d(CAXYAGCACGAC),
where the underlined XY represents either TT, T[]T, TC,
TmCm or T[]mCm, were hybridized to dN-ended 9-mers,
d(GTCGTGCTN) (4 nmol each), in a buffer containing
10 mM phosphate, 100 mM NaCl and 0.1 mM EDTA,
pH 7.0 (50 ml), by heating at 65ꢃC for 5 min and cooling
to room temperature overnight. Under the annealing con-
ditions, ꢀ5% of T[]mCm was hydrolyzed to T[]T. Each of
the duplex solutions was diluted with the same buffer to
total concentrations (Ct) of 4.27, 5.97, 8.53, 11.95 and
17.1 mM. Melting curves were measured at 260 nm on a
Shimadzu PharmaSpec UV-1700 UV/Vis spectrophotom-
eter equipped with a TMSPC-8 temperature controller
module. The temperature was raised from 18 to 70ꢃC at
a rate of 0.5ꢃC minꢂ1, and the Tm values were obtained by
the two-point average method, using the data processing
software supplied by the manufacturer. The measurements
were repeated three times, and the averaged Tm values
were used for the following analyses.
To obtain the thermodynamic parameters, a van’t
Hoff analysis was performed, according to the previous
report (26). The reported equation 1/Tm = R/ÁHꢃ ln

Nucleic Acids Research, 2014, Vol. 42, No. 3 2079
i and ii were treated with TEA 3HF and thiophenol in
were verified, to prepare its phosphoramidite building
block. The dinucleoside monophosphate of the cis-syn
T[]mCm (9) was quite stable to treatments with 80%
CH3COOH for 1 h or 28% ammonia water for 2 h at
ambient temperature (Supplementary Figure S4), which
simulated the detritylation and ultra-mild deprotection
conditions in the oligonucleotide synthesis, respectively.
To test the reactivity with the capping reagents in the
oligonucleotide synthesis, we used the abundant
diastereomer of trans-syn (SYN-ANTI) T[]mCm (5), to
save the cis-syn isomer. The isolated trans-syn isomer of
compound 5 (peak i in Figure 2) was treated with excess
amounts of phenoxyacetic anhydride in the presence of
1-methylimidazole, to determine whether the methylamino
group could be acylated with the capping reagents.
Considering the steric hindrance at the reactive amidine
groups in the stereoisomers of compound 5, the cis-syn
T[]mCm was expected to be inert to the reaction if the
trans-syn T[]mCm, which contains less hindered amidine,
was not acylated. After 2 h, the product was purified by
silica-gel column chromatography and was treated with
28% ammonia water for 2 h. After these treatments, the
intact trans-syn T[]mCm (5) was retained, indicating that
the methylamino group of the trans-syn T[]mCm did not
react with the capping reagents. These comprehensive
results suggested that T[]mCm is chemically stable to the
reactions in the oligonucleotide synthesis. After 50-protec-
tion, 30-deprotection and 30-phosphitylation, the building
block of the cis-syn T[]mCm (8) was successfully
synthesized (Scheme 2).
·
turn, to remove the protecting groups. We found that the
deprotection of the products (peaks i and ii) gave the same
product (data not shown), indicating that peak ii was the
other trans-syn (SYN-ANTI) diastereomer caused by the
chiral phosphotriester group. The products yielding peaks
iii and iv were treated with TEA 3HF and thiophenol
·
in the same manner, and their deprotection products
were found to be identical to each other, but not to that
obtained from the compounds yielding peaks i and ii (data
not shown). After HPLC purification of this deprotection
product (referred to as compound 9), its NMR and mass
spectra were measured. In the NOESY spectrum of
compound 9 measured in D2O, correlations of H20–H6
in both the 50 and 30 components were observed
(Supplementary Figure S3), indicating that its stereochem-
istry was cis-syn with the ANTI-ANTI conformation.
Since the mass analysis of compound 9 also supported
the presence of the methylamino group, peaks iii and iv
were the Sp and Rp diastereomers of the cis-syn (ANTI-
ANTI) structure of compound 5. Fortunately, one of the
cis-syn diastereomers (peak iv) was purified by silica-gel
column chromatography, in the best isolated yield of
7.3%, and was characterized by NMR spectroscopy and
mass spectrometry. Since the yield was not high, we
repeated the 2 h irradiation of compound 4 six times, to
compensate for the low yield, and finally the cis-syn isomer
of compound 5 was obtained in an amount sufficient for
the oligonucleotide synthesis.
Next, the chemical stability and reactivity of the
methylamino group within the obtained CPD (T[]mCm)
Synthesis and characterization of the oligonucleotides
containing T[]mCm
A 12-mer oligonucleotide containing the cis-syn T[]mCm,
d(CGTACT[]mCmCATGC), was synthesized on a DNA
synthesizer, and was purified by HPLC. The ESI-LC mass
spectrometry revealed that the molecular weight of the
Figure 2. HPLC analysis of the cycloaddition reaction of N-methylated
dinucleoside monophosphate (4) in the presence of photosensitizer,
monitored at 230 nm. Aliquots of the reaction mixture were analyzed
after UV irradiation for 0 (a), 0.5 (b), 1 (c), 1.5 (d) and 2 h (e).
Scheme 2. Synthesis of the phosphoramidite building block of the
cis-syn isomer of T[]mCm. Reagents and conditions: (a) DMTCl/
pyridine, (b) TEA 3HF/THF and (c) 2-cyanoethyl N,N-diisopropyl-
·
chlorophosphoramidite, ethyldiisopropylamine/THF.

2080 Nucleic Acids Research, 2014, Vol. 42, No. 3
purified oligonucleotide was identical to the calculated
value, demonstrating the presence of the methylamino
group (Supplementary Figure S1). This result also sup-
ported that the cis-syn T[]mCm was inert to the capping
reaction. The oligonucleotide was characterized further by
UV irradiation and an enzymatic reaction. Both the UV
irradiation of the T[]mCm 12-mer and the treatment with
Escherichia coli CPD photolyase yielded a single product,
which co-eluted with the TmCm 12-mer (Supplementary
Figure S5). Although the desired product was obtained,
we detected an extra peak, in addition to that of the
desired product, in the HPLC analysis after the purifica-
tion of the T[]mCm 12-mer (Supplementary Figure S5,
trace a). The newly emerged minor peak co-eluted with
an oligonucleotide containing the cis-syn TT-CPD
(T[]T), suggesting that hydrolysis occurred at the
methylamino group. We found that the hydrolysis was
caused by the repeated coevaporations with water to
remove TEAA after the HPLC purification.
Since the hydrolyzed product was detected in the HPLC
analysis, the thermal stability of the methylamino group
within T[]mCm to hydrolysis was investigated in detail. A
solution of the T[]mCm 12-mer, dissolved in phosphate
buffer at pH 7.4, was incubated at 50, 80 and 95ꢃC, and
aliquots were analyzed by HPLC at appropriate intervals.
Upon the incubation, the T[]mCm 12-mer was converted
into the T[]T 12-mer. The logarithm of the decreasing
fraction of T[]mCm 12-mer was plotted against the incuba-
tion time, and these data were fitted with a pseudo-first
order equation to obtain the observed rate constants
(kobs) (Supplementary Figure S6A). From kobs, the half-
lives (t1/2) were calculated, as shown in Table 1. The t1/2
of T[]mCm at 50ꢃC was found to be 51 h, and extrapolation
of our results indicated that its half-life under physiological
conditions (37ꢃC at pH 7.4) was 176 h, i.e. >7 days. From
the Arrhenius plot of the obtained kobs (Supplementary
Figure S6B), the activation energy of hydrolysis (Ea) and
the frequency factor (A) were determined as 77.7 kJ molꢂ1
and 7.97 ꢁ 108 minꢂ1, respectively.
16-nucleotide (nt) primer hybridized to the TT template,
no extended products were observed for the templates
containing the cis-syn T[]T and cis-syn T[]mCm
(Figure 3B, lanes 1–5 and 10–12), indicating that
T[]mCm blocks chain elongation by replicative polymer-
ases, as demonstrated for T[]T. hPolZ, which is respon-
sible for the error-free bypass of T[]T (lanes 6–8 in
Figure 3B), successfully bypassed T[]mCm and extended
the primer to the 30-mer, although the polymerase was
considerably stalled at the site opposite the 30 component
of T[]mCm (lanes 13–15).
To determine which nucleotides are incorporated
opposite T[]mCm, the primer extension analysis was per-
formed in the presence of each dNTP. hPolZ mainly
incorporated dGMP and TMP opposite the mCm moiety
of T[]mCm, as the 17th nucleotide of the primer
(Figure 3C). To investigate which nucleotide was prefer-
entially incorporated, kinetic analysis of the incorporation
was performed under single-completed hit conditions.
The initial velocities of the reactions were well-fitted
with the Michaelis–Menten equation (Supplementary
Figure S7), and the catalytic rate constants (kcat) and the
Michaelis constants (Km) were obtained by non-linear
least squares regression (Table 2). A comparison of the
efficiencies, namely kcat/Km, clearly showed that the in-
corporation of TMP was unexpectedly efficient, and just
1.7-fold less frequent than that of dGMP. These results
suggested that hPolZ slightly preferred to incorporate
dGMP as the correct nucleotide opposite the mCm
moiety of T[]mCm, but also incorporated the incorrect
TMP at a relatively high efficiency.
Since the misincorporation of TMP at the 17th position
of the primer could block further extension of the primer,
the analysis using 17-nt templates containing dGMP or
TMP at the 30 end (dG17 or T17, respectively) was per-
formed. As expected, almost no extended product was
observed for T17, whereas hPolZ extended the dG17
primer and incorporated dAMP opposite the 50 compo-
nent of T[]mCm (Figure 3D). These results indicated that
TLS across T[]mCm could occur in an error-free manner
once hPolZ incorporated dGMP opposite the mCm of
T[]mCm, but more than 30% of the primer extension
stopped at the site opposite the mCm moiety, due to the
misincorporation of TMP.
Translesion synthesis past T[]mCm by human DNA
polymerase g (hPolg)
A longer oligonucleotide, T[]mCm 30-mer, was synthesized
to investigate the biochemical properties of T[]mCm, and
primer extension analysis was performed using KF, which
is deficient in the exonuclease activity, and human DNA
polymerase Z (hPolZ). While KF extended the 32P-labeled
Thermodynamic analysis of the formation of duplexes
mimicking the primer-template structures
To investigate the mispairing ability of the mCm moiety of
T[]mCm with TMP, a thermodynamic analysis was per-
formed using short oligonucleotides mimicking the
primer-template structure. The 12-mer oligonucleotides,
d(CAXYAGCACGAC), where XY represents TT, TC,
TmCm, T[]T or T[]mCm, were hybridized to the dN-
ended 9-mer oligonucleotides, d(GTCGTGCTN),
and the thermodynamic parameters for the duplex forma-
tion were obtained by the van’t Hoff plot of the melting
temperatures (Supplementary Figure S8; Table 3). In this
system, the 12-mers and 9-mers mimic the templates
and primers, respectively, and therefore, the differences
between the obtained parameters could be used to
Table 1. Half-lives (t1/2) and Arrhenius parameters of T[]mCm and
T[]mC hydrolysis in the oligonucleotides
Half-lives (t1/2) (h)
Arrhenius parameters
37ꢃC
50ꢃC
80ꢃC
Ea (kJ molꢂ1
)
A (minꢂ1
)
T[]mCm a
T[]mC
176 b
8.25 c
51.3
4.8
77.7
86.5
7.97 ꢁ 108
5.16 ꢁ 1011
2.13 c
0.14 b
aThis work.
bThese values were estimated by extrapolation with the Arrhenius plot
in Supplementary Figure S6.
cHalf-lives for T[]mC at 37 and 50ꢃC were extracted from (17).

Nucleic Acids Research, 2014, Vol. 42, No. 3 2081
Figure 3. (A) Structure of the 32P-labeled 16-mer primer and the 30-mer templates for TLS by hPolZ. XY in the template sequence represents TT,
cis-syn T[]T or cis-syn T[]mCm. (B) Primer extension assays using KF and hPolZ. The primer-templates containing the cis-syn T[]T (lanes 2–8) and
cis-syn T[]mCm (lanes 9–15) were incubated at 37ꢃC with increasing amounts of KF (0.0125, 0.025, 0.1 units) or hPolZ (8, 32, 128 fmol) for 15 min.
The samples for lanes 3 and 9 contained no polymerases. For the positive control, the primer-template containing normal TT (lane 1) was also
incubated with KF (0.1 units) at 37ꢃC for 15 min. (C) Primer extension assays of the cis-syn T[]mCm (lanes 1–5) and cis-syn T[]T (lanes 6–10) in the
presence of hPolZ and each dNTP. The primer-templates were incubated with hPolZ (8 fmol) at 37ꢃC for 5 min. (D) Primer extension assays using
dG17 (lanes 1–5) or T17 (lanes 6–10) primers in the presence of hPolZ and each dNTP. The primer-templates were incubated with hPolZ (16 fmol) at
37ꢃC for 5 min.
Table 2. Kinetic parameters for nucleotide incorporation opposite the
mCm moiety of T[]mCm
Table 3. Thermodynamic parameters for duplex formation
Duplex
ÁHꢃ
ÁSꢃ
ÁGꢃ
Tm
(Ct = 12.0 mM)
(kcal molꢂ1
)
(cal molꢂ1 Kꢂ1
)
(kcal molꢂ1 a
)
Substrates
kcat (minꢂ1
)
Km (mM)
kcat/Km
(minꢂ1 mMꢂ1
Efficiency
)
TTꢄA
ꢂ84.5 1.5
ꢂ63.8 0.6
ꢂ54.9 0.1
ꢂ52.1 0.1
ꢂ80.4 0.1
ꢂ56.8 0.2
ꢂ48.6 0.4
ꢂ53.3 0.3
ꢂ79.4 0.5
ꢂ93.3 0.4
ꢂ68.9 1.0
ꢂ66.6 0.6
ꢂ59.5 0.02
ꢂ74.2 0.7
ꢂ59.1 0.3
ꢂ58.5 0.3
ꢂ65.7 0.8
ꢂ92.1 0.8
ꢂ68.0 0.5
ꢂ85.0 0.2
ꢂ242 4.4
ꢂ177 1.6
ꢂ149 0.3
ꢂ140 0.2
ꢂ229 0.4
ꢂ155 0.6
ꢂ129 1.1
ꢂ144 0.9
ꢂ227 1.5
ꢂ265 1.1
ꢂ194 2.8
ꢂ187 1.7
ꢂ164 0.06
ꢂ211 2.1
ꢂ163 0.8
ꢂ161 0.8
ꢂ183 2.3
ꢂ267 2.3
ꢂ191 1.4
ꢂ245 0.7
ꢂ12.3 0.08
ꢂ11.0 0.02
ꢂ10.5 0.006
ꢂ10.3 0.003
ꢂ12.0 0.01
ꢂ10.7 0.01
ꢂ10.0 0.02
ꢂ10.3 0.01
ꢂ11.8 0.03
ꢂ14.2 0.03
ꢂ11.1 0.04
ꢂ10.9 0.03
ꢂ10.4 0.001
ꢂ11.4 0.04
ꢂ10.5 0.01
ꢂ10.5 0.01
ꢂ11.0 0.04
ꢂ12.6 0.04
ꢂ11.1 0.03
ꢂ12.1 0.01
42.9
42.3
41.8
41.8
42.7
42.3
41.2
41.1
42.1
48.0
41.0
40.7
40.2
41.3
40.8
40.9
41.4
42.3
41.6
41.7
TTꢄG
dGTP
TTP
121 12
172 20
18.7 5.3
44.3 11.2
6.47 1.94
3.88 1.08
1
0.6
TTꢄC
TTꢄT
T[]TꢄA
T[]TꢄG
T[]TꢄC
T[]TꢄT
TCꢄA
discuss the mechanism of nucleotide incorporation
by hPolZ.
TCꢄG
TCꢄC
TCꢄT
Among the TTꢄN duplexes, TTꢄA was the most stable as
expected, and its ÁGꢃ was ꢂ12.3 kcal molꢂ1 (Figure 4A;
Table 3). Upon CPD formation, the ÁGꢃ for T[]TꢄA was
slightly increased by ꢀ0.3 kcal molꢂ1, but the 30 compo-
nent of T[]T can form a stable base pair with adenine. The
TC-containing 12-mer formed a stable duplex with the
TmCmꢄA
TmCmꢄG
TmCmꢄC
TmCmꢄT
T[]mCmꢄA
T[]mCmꢄG
T[]mCmꢄC
T[]mCmꢄT
dG-ended 9-mer, and its ÁGꢃ was ꢂ14.2 kcal molꢂ1
,
which is 2 kcal molꢂ1 less, as compared with the other
nucleobases. The most stable duplex among the
TmCmꢄN duplexes was the TmCmꢄG, but its selectivity
for G was much reduced compared to that of TC,
probably because the cis-rotamer of mCm inhibited the
formation of the Watson–Crick mCmꢄG base pair
(31,32). In the case of T[]mCm, however, the stability of
the mCmꢄT pair was increased upon CPD formation, and
its ÁGꢃ was ꢂ12.1 kcal molꢂ1. The most stable duplex
among the T[]mCmꢄN duplexes was T[]mCmꢄG, with a
ÁGꢃ of ꢂ12.6 kcal molꢂ1, and the reduced DGꢃ value
compared to that for TmCmꢄG suggested that the mCm
aAt 25ꢃC.
moiety of the T[]mCm likely formed the Watson–Crick
base pair with guanine (Figure 4B). These results sug-
gested that the N-methyl group drastically changes the
base-pairing ability of cytosine, and that the mCm
moiety of T[]mCm can form a thermodynamically stable
base pair with thymine, which causes the misincorporation
of TMP opposite T[]mCm by hPolZ.

2082 Nucleic Acids Research, 2014, Vol. 42, No. 3
To compare the thermal stabilities of T[]mCm and
T[]mC, the Ea and A values for the hydrolysis of T[]mC
were determined, using the kinetic parameters reported by
Song et al. (17) (Table 1). It should be noted that a certain
level of error may be included in the following values due
to the differences in the experimental conditions, because
several factors, such as ionic strength of the solution (34)
and the nucleotides flanking the lesion (35), significantly
influence the deamination rate. Ea values in Table 1,
apparently suggested that the N-methyl modification
reduced the stability to hydrolysis. The hydrolytic deamin-
ation of cytosine is reportedly accelerated under acidic
conditions (34), and the probability of the protonation
of the N3 position of the mCm moiety would be increased,
due to the electron-donating ability of the N-methyl
group. This factor should have increased the sensitivity
to temperature and reduced the activation energy. While
the values of the activation energy were similar, the fre-
quency factor was 650-fold smaller than that for T[]mCm.
This accounts for the strong resistance of T[]mCm to hy-
drolysis. The N-methylation of the exocyclic amino group
directly reduces the hydrolysis frequency, due to its
electron-donating ability. Vilkaitis and Klimasauskas
reported a bisulfite sequencing method that displays N4-
methylcytosine, as well as mC (20). Although they did not
discuss its mechanism, it can now be explained by the
enhanced stability of the bisulfite-attached intermediate
to hydrolysis, in a similar manner to the case of T[]mCm.
T[]mCm blocked DNA synthesis by the replicative DNA
polymerase, in the same manner as T[]T (Figure 3B). Since
the stalled polymerases can be replaced with TLS poly-
merases in cells, the bypass of T[]mCm by Y-family
DNA polymerases was investigated. In this study, we
chose human DNA polymerase Z as the TLS polymerase,
to evaluate the effect on the N-methyl modification by
comparing the results between this and previous (17)
studies. Our biochemical analysis revealed that human
DNA polymerase Z preferentially incorporated dGMP
opposite the mCm moiety of T[]mCm, although TMP
was also considerably incorporated. The efficiency of
TMP incorporation opposite the mCm moiety was 60%
of that of dGMP, and TMP incorporation strongly
blocked further chain elongation. hPolZ reportedly
incorporated incorrect nucleotides opposite the cisplatin-
induced intrastrand crosslinks between two guanines and
N-2-acetylaminofluorene-modified guanine (AAF-G) (6).
In the case of AAF-G, the kinetic analysis was performed.
The efficiencies of incorrect nucleotide incorporation were
3–4% in total, and the incorporated incorrect nucleotides
severely blocked the subsequent primer extension (25). As
compared to these results, the misincorporation of TMP
opposite T[]mCm occurred quite frequently. Yeast and
human DNA polymerases Z reportedly incorporated
guanine opposite the mC moiety of T[]mC, and the
misincorporation of adenine was observed at only one
thirty-second of the frequency, as compared to the incorp-
oration of guanine (17), suggesting that the N-methyl
group would drastically change the base-pairing ability
of the mC moiety.
Figure 4. (A) Comparison of the thermodynamic stabilities of the base
pairs at the 30 component of CPD. Absolute values of Gibbs free
energies (Table 3) are shown. (B and C) Plausible base pair formation
by the mCm moiety of T[]mCm with guanine (B) and thymine (C).
DISCUSSION
We synthesized partially protected thymidylyl-(30!50)-
20-deoxy-N4,5-dimethylcytidine and performed its phot-
osensitized [2+2] photocycloaddition, which resulted
in the formation of the CPD between thymine and
N4,5-dimethylcytosine (T[]mCm), without significant
hydrolytic deamination. However, in this reaction, the
trans-syn isomer, which is produced much less efficiently
than the cis-syn CPD upon UV irradiation of isolated
DNA (33), was the major product, in contrast to the
low yield of the cis-syn isomer. Consistent with our
previous report on the stereoselective CPD formation
(18), the modification of the exocyclic amino group sig-
nificantly affected the stereoselectivity of CPD formation,
and the relative yield of the cis-syn isomer was lowered,
due to the steric hindrance by the N-modification group.
We also tested the photocycloaddition of thymidylyl-
(30!50)-20-deoxy-N4-methylcytidine
phosphotriester,
which lacked the C5 methyl group within the mCm
moiety of compound 4. In this case, the cis-syn CPD
(T[]Cm) was hardly detected, and the trans-syn isomer
was produced predominantly, while the reaction using
compound 4 gave the cis-syn T[]mCm. This is the reason
why we used mC. Notably, the trans-syn T[]Cm was also
stable enough to be purified as the intact form. This result
indicated that the N-methyl modification of cytosine,
rather than the C5 methyl group, was important for the
resistance to hydrolysis.
The thermodynamic analysis of the duplexes mimicking
the primer-template structure revealed that T[]mCm

Nucleic Acids Research, 2014, Vol. 42, No. 3 2083
formed a stable base pair with thymine, and its stability was
slightly lower than that of the T[]mCmꢄG pair (Figure 4A).
This result is in good agreement with our biochemical
kinetic analysis of the incorporation efficiencies, and the
misincorporation due to the mismatch formation can also
be explained by the mechanistic study of hPolZ in crystals
(8). It is reported that an incoming nucleotide forms a base
pair with the template nucleotide at a ground state prior to
making the phosphodiester bond, and therefore once the
incoming nucleotide and the other factors are
accommodated properly in the active site, nucleotide in-
corporation can occur. The slightly less efficient incorpor-
ation of TMP than that of dGMP by hPolZ would be
caused by the different stabilities of the base pairs. The
mCm moiety of T[]mCm can form a canonical Watson–
Crick base pair with guanine, in which the methyl group
protrudes into the major groove (Figure 4B). To discuss the
T[]mCmꢄT pair, a model structure showing the incorpor-
ation of TMP opposite the 30 component of the T[]T, in
which the incoming TMP was fitted to AMPNPP, was con-
structed by using the reported ternary complex structure
(Supplementary Figure S9) (7). Since the O4 of the
incoming TMP is located close to the N4 of AMPNPP,
the O4 of TMP may be involved in hydrogen bond forma-
tion with the N4 of the mCm moiety of T[]mCm. However,
the N3 of the mCm moiety is too far away to form a
hydrogen bond with the N3 of TMP. Since the ÁGꢃ of
the T[]mCmꢄT pair is comparable to that of the T[]mCmꢄG
pair (Figure 4A), it is unlikely that only one hydrogen bond
is formed between the 30 component of T[]mCm and TMP,
in the geometry shown in Supplementary Figure S9. Taking
into account the incorporation of TMP by hPolZ, the CPD
in the template strand should be rotated in the direction
shown by the arrow, and the tautomerization of T[]mCm
would enable the N3 and the O2 of the 30 component of
T[]mCm to form hydrogen bonds with the O4 and the N3 of
TMP, respectively (Figure 4C). Since the 50 and 30 compo-
nents of T[]mCm are bridged via the cyclobutane ring, the
shifted position of the mCm moiety causes the incorrect
positioning of the 50 component of T[]mCm, resulting in
the blockage of further primer extension.
In conclusion, we performed the photocycloaddition
reaction of thymidylyl-(30!50)-20-deoxy-N4,5-dimethyl-
cytidine, and found that the obtained CPD, T[]mCm,
was quite stable to hydrolysis. Kinetic analyses indicated
that the electron-donating N-methyl group reduces the
frequency of T[]mCm hydrolysis. Biochemical analyses
revealed that T[]mCm could be bypassed correctly by
human DNA polymerase Z, but the incorporation of the
incorrect TMP caused a severe blockage of DNA synthe-
sis. The N-methylation of cytosine is a simple modifica-
tion, but unexpectedly modulated the chemical and
biochemical properties of cytosine-containing CPDs. N4-
methylcytosine has been found in bacterial genomes as a
minor base (19,36), but has not been detected in eukary-
otes thus far, despite the development and improvement of
analytical instruments and techniques. Since thermophilic
bacteria also have putative Y-family DNA polymerases,
such as the DinB and UmuC proteins (37), TLS by these
proteins will be investigated in the future.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
FUNDING
Management expenses grants from the Ministry of
Education, Culture, Sports, Science and Technology,
Japan. Funding for open access charge: Management
expenses grants from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
Conflict of interest statement. None declared.
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