2-Pyrimidinone as a probe for studying the
Eco
RII DNA
methyltransferase–substrate interaction
Oksana M. Subach
1
, Anton V. Khoroshaev
1
, Dmitrii N. Gerasimov
1
, Vladimir B. Baskunov
1
,
Anna K. Shchyolkina
2
and Elizaveta S. Gromova
1
1
Chemistry Department, Moscow State University, Russia;
2
Engelghardt Institute of Molecular Biology, Russian Academy of
Sciences, Moscow, Russia
EcoRII DNA methyltransferase (M.EcoRII) recognizes
the 5¢…CC*T/AGG…3¢ DNA sequence and catalyzes the
transfer of the methyl group from S-adenosyl-
L
-methionine
to the C5 position of the inner cytosine residue (C*). Here,
we study the mechanism of inhibition of M.EcoRII by DNA
containing 2-pyrimidinone, a cytosine analogue lacking an
NH

DNA (cytosine-5)-methyltransferases (C5 MTases) catalyze
the transfer of a methyl group from S-adenosyl-
L
-methio-
nine (AdoMet) to cytosine C5 atom in specific DNA
sequences. The methylation reaction of C5 MTases occurs
with the addition of a cysteine thiol group from the
conserved Pro-Cys motif to the C6 position of the target
cytosine, followed by methyl transfer from AdoMet to the
C5 position of the target base and the release of the
methylated substrate [1,2] (Fig. 1A). It is important to note
that the target cytosine is flipped out of the DNA double
helix into the catalytic pocket of the enzyme and brought
into proximity of the cofactor [2].
Several cytosine analogues, 5-fluorocytosine (FC),
5-azacytosine (AzaC) and 2-pyrimidinone (2P), have been
reported as mechanism-based inhibitors of C5 MTases
[3–6]. Introduction of a fluorine atom to the C5 position of
the target cytosine results in an irreversible covalent attack
of a cysteine residue and transfer of a methyl group to the
C5 position of the target base [1]. Replacement of C5 by a
nitrogen atom in azacytosine (AzaC) facilitates nucleophilic
attack of the cysteine residue at the C6 position which
occurs in the presence or absence of AdoMet [5]. Methyl or
proton transfer to the N5 position occurs in the presence or
absence of AdoMet, accordingly. As a result, two structures
of the end product are possible: the enzyme linked to
methylated AzaC or the enzyme linked to protonated AzaC
[5].AsthereisnoprotonatC5totakeawaywhentheN5
position becomes methylated an irreversible covalent com-

(Received 18 November 2003, revised 19 February 2004,
accepted 14 April 2004)
Eur. J. Biochem. 271, 2391–2399 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04158.x
presence of AdoMet was not investigated. It should be
noted that 1-(b-
D
-ribofuranosyl)-2-pyrimidinone, often
referred to as zebularine, was used in vivo as an antitumor
drug [10]. By now it is clear that its antitumor properties are
likely attributed to inhibition of C5 MTase activity in tumor
cells. 2-Pyrimidinone could also be considered as a mimic
of an intermediate in the minor deaminative pathway of
C5 MTases catalysis [6].
EcoRII DNA methyltransferase (M.EcoRII) catalyzes
the transfer of a methyl group from AdoMet to the C5
position of the inner deoxycytidine residue (C*) in the DNA
sequence 5¢…CC*T/AGG…3¢. It has been shown that
M.EcoRII forms an irreversible covalent complex with
DNA containing FC instead of the target cytosine [11,12].
Introduction of AzaC in this position also led to inhibition
of the enzyme [5]. The mechanism of inhibition of M.Eco-
RII by 2P-containing DNA is still unknown. Also, a study
of the contributions of different functional groups of the
5¢…CCT/AGG…3¢ sequence to specific interaction with
M.EcoRII is at the very beginning [13].
We aim to explore the effect of 2P substitution for C and
T within the recognition sequence of M.EcoRII on recog-
nition and catalysis performed by M.EcoRII. In this work,
we have examined a DNA duplex containing 2-pyrimidi-
none instead of the target cytosine as a potential inhibitor

T4 polynucleotide kinase was from MBI Fermentas
(Vilnius, Lithuania).
Oligonucleotides
Oligodeoxyribonucleotides containing 2-pyrimidinone
were synthesized as described previously [15].
32
P-phosphory-
lation of the oligonucleotides was performed using
T4-polynucleotide kinase and [
32
P]ATP[cP].
UV thermal denaturation and thermodynamic parameters
of duplex formation
Heating of the samples containing 2.25 l
M
of duplexes in
buffer A (40 m
M
Tris/HCl, pH 7.9, 1 m
M
EDTA, 50 m
M
NaCl) at temperatures ranging from 15 to 85 °Cwas
performed at a constant rate of 0.2 °CÆmin
)1
. Absorbance
of duplexes at 260 nm was measured using Cary 50 Bio
spectrophotometer (Varian, Victoria, Australia) with tem-
perature controller. Thermodynamic analysis of helix-coil
transition curves was performed using a two-state model.

Fig. 1. Proposed mechanism of inhibition of M.EcoRII by 2P-containing DNA duplexes in the absence [6] (A) or in the presence (B) of AdoMet. In the
case of M.EcoRII, the amino acid residue attacking the C6 position is Cys186 [11,12]; the general acid donating a proton to N3 is probably Glu233
[4,28].
2392 O. M. Subach et al. (Eur. J. Biochem. 271) Ó FEBS 2004
were analysed by 8% native PAGE for 3 h at 120 V. The gel
was prerun for 1 h at 100 V. Autoradiographs of the gels
were prepared using Molecular Dynamics Phosphorimager
(Amersham Biosciences, USA). Radioactivities of M.Eco-
RII–DNA complex (cpm
bound
) and free DNA (cpm
free
)
were determined. The ratio of bound to free DNA was
calculated as (cpm
bound
)/(cpm
free
); the concentration of
bound DNA was calculated as [S
0
][(cpm
bound
)/(cpm
bound
+
cpm
free
)]. Data were analyzed by linear regression using the
Microcal

determined and the fraction of bound DNA was calculated
as (cpm
bound
)/(cpm
bound
+cpm
free
). K
app
d
was calculated by
fitting the data to the following equation derived from a
standard bimolecular binding equilibrium as described [16]:
cpm
bound
ðcpm
bound
þcpm
free
Þ
¼
½ES
½S
0

¼
A
2½S
0


M.EcoRII concentration; A is the factor accounting for
nonideal equilibrium conditions during electrophoresis
(cage effect, thermal dissociation). Nonlinear regression
was performed using Microcal
ORIGIN
6.0 software.
Detection of DNA-enzyme adducts
All reactions of
32
P-labeled duplexes I–III (100 n
M
)with
M.EcoRII (200 n
M
) were performed in 15 lL of buffer B
containing 0.5 m
M
AdoMet or 0.5 m
M
AdoHcy. Reaction
mixtures were incubated for 30 min at room temperature
andfor15minoniceandprocessedbyoneofthefollowing
ways: (a) incubated with 1% SDS at room temperature for
10 min and analyzed by 8% native PAGE; (b) incubated
with 1% SDS at 65 °C for 5 min and analyzed by 8% native
PAGE; (c) incubated with 0.8% SDS at room temperature
for 10 min and analyzed by 12% SDS/PAGE (Laemmli
gel).
For autoradiography of the electrophoretic pattern,
Kodak-XOMAT-S film was exposed with an intensifier

radioactivity was measured on Tracor Analytic Delta 300
scintillation counter (ThermoQuest/CE Instruments,
Piscataway, USA) and the amount of methylated DNA
was determined as described [17]. Data were analyzed by
linear regression using the Microcal
ORIGIN
6.0 software.
For quantification of the transfer of methyl groups to a
2-pyrimidinone residue in DNA duplexes by M.EcoRII
methylation reactions were performed in 10 lL of buffer B,
containing duplexes I, Im, III, IIIm or V (500–1000 n
M
),
M.EcoRII (12.5–1000 n
M
)and[CH
3
–
3
H]-AdoMet
(1.3 l
M
). In the case of M.HhaI methylation reactions were
performed in 10 lL of buffer C (50 m
M
Tris/HCl, pH 7.5,
5m
M
2-mercaptoethanol, 10 m
M

3
H]-AdoMet (1.3 l
M
) in buffer
Bat15°C for the indicated time periods. Reaction mixtures
(8 lL) were pipetted onto DE81 (Whatman) paper disks
and processed as described for determination of V
0
.
Results and discussion
To elucidate the mechanism of inhibition of M.EcoRII
by 2-pyrimidinone modified DNA, and to understand the
role of functional groups of pyrimidine bases of the
recognition sequence in specific DNA–M.EcoRII inter-
action, a series of 2P-containing substrate analogues
was synthesized (duplexes II–IV, Table 1 and duplexes
Iim–IVm, Table 2).
Thermodynamics of formation of the
2P
-containing DNA
Insertion of 2P in place of C or T resulted in a marked
destabilization of DNA duplexes [18–21]. To ascertain
whether the 2P-containing DNA duplexes II–IV had a
double helix structure under conditions of methylation by
M.EcoRII, the thermodynamic stability of these duplexes
was evaluated (Table 1). The values of the free energy of
transition, as well as of the transition temperature (melting
temperature, T
m
), point to the following duplex stabilities:

the conservative CD spectrum in comparison with the
contributions of cytosine and thymine. Thus, the observed
minor dissimilarity in the CD spectra of duplexes II–IV
from CD spectrum of duplex I can be attributed to the
distinctive optical features of the 2P analog, rather than to a
marked distortion of the DNA conformation.
Determination of concentration of active form
of M.EcoRII
It is known that the concentration of the active form of
DNA methyltransferases does not correspond accurately to
the total protein concentration [5,22]. The concentration of
active form of M.EcoRII was estimated by titration of the
enzyme (20 n
M
) with M.EcoRII substrate in the presence of
AdoHcy (Fig. 3, inset). The ratio of bound to free DNA
was plotted vs. concentration of bound DNA (a Scatchard
plot, Fig. 3) [23]. An 18-mer DNA duplex 5¢-GAG
CCAACCTGGCTCTGA-3¢/3¢-CTCGGTTGGACCGAG
ACT-5¢ (I¢) was used as M.EcoRII substrate. The horizontal
axis intercept gives the total concentration of DNA binding
sites (n[E
0
]) equal to 2.35 ± 0.12 n
M
(Fig. 3) or
2.04 ± 0.25 (data not shown). As the number of DNA
binding sites per molecule of enzyme (n) is 1 for M.EcoRII,
the average concentration of M.EcoRII active form is
2.2 ± 0.2 n

Æmin
)1
)V
rel
0
(%)
Im
5¢-GCCAACCTGGCTCT-3¢/ 172.0 ± 34.4 100 ± 20
3¢-CGGTTGGAMCGAGA-5¢
IIm
5¢-GCCAA
2P
CTGGCTCT-3¢/ 197.8 ± 53.3 115 ± 31
3¢-CGGTT-GGAMCGAGA-5¢
IIIm
5¢-GCCAAC
2P
TGGCTCT-3¢/0 0
3¢-CGGTTG-GAMCGAGA-5¢
IVm
5¢-GCCAACC
2P
GGCTCT-3¢/ 34.4 ± 3.4 20 ± 2
3¢-CGGTTGG-AMCGAGA-5¢
Fig. 2. CD spectra of DNA duplexes I–IV. ––,I;–d–, II; –m–, III;
–j–, IV. Difference of CD spectra of I and duplexes: –s–, II; –n–, III;
–h–, IV. Temperature was 20 °C.
Table 1. Thermodynamic parameters of formation of 2P-containing DNA duplexes determined from thermal denaturation curves. Thermodynamic
parameters and their standard deviations were determined from fitting the theoretical melting curves to experimental curves (see Materials and
methods). Standard deviation of DS was less than 0.1 calÆmol

5¢-GCCAACCTGGCTCT-3¢/ 52.6 ± 0.1 )65.0 ± 0.6 )173 )14.3 ± 0.6 –
3¢-CGGTTGGACCGAGA-5¢
II 5¢-GCCAA
2P
CTGGCTCT-3¢/ 46.0 ± 0.1 )43.9 ± 0.7 )111 )11.5 ± 0.7 2.8
3¢-CGGTT-GGACCGAGA-5¢
III
5¢-GCCAAC
2P
TGGCTCT-3¢/ 41.4 ± 0.1 )36.3 ± 0.4 )88 )10.3 ± 0.4 4
3¢-CGGTTG-GACCGAGA-5¢
IV
5¢-GCCAACC
2P
GGCTCT-3¢/ 35.4 ± 0.1 )41.7 ± 0.7 )106 )9.9 ± 0.7 4.4
3¢-CGGTTGG-ACCGAGA-5¢
2394 O. M. Subach et al. (Eur. J. Biochem. 271) Ó FEBS 2004
binding and methylation of canonical (I) and the 2P-
containing DNA duplexes (II-IV) with M.EcoRII (Table 3).
The formation of complexes was monitored by gel mobility-
shift assays in the presence of AdoHcy because of the
known increase in the affinity of M.EcoRII for DNA in the
presence of the cofactor [12]. DNA duplexes were incubated
with increasing M.EcoRII concentrations at saturating
AdoHcy concentrations. A binding isotherm and corres-
ponding autoradiograph of a typical experiment are shown
in Fig. 4. The calculated apparent dissociation constants
(K
app
d

the target cytosine with a K
app
d
similar to that of duplex I
(Table 3). It could be that the 4-NH
2
group of the target
cytosine is not essential for recognition of DNA by
M.EcoRII as was suggested for several other MTases, as
base flipping probably occurs with any base at the target
position [26]. However, in the case of the M.EcoRII complex
with duplex III such a simple conclusion is ambiguous
because in addition to the noncovalent complex a stable
Fig. 3. Scatchard plot of the ratio of bound to
free DNA substrate vs. concentration of bound
DNA substrate. Inset: autoradiograph of gel
shift assay of M.EcoRII with DNA substrate
I¢. Lanes 1–4: 20 n
M
M.EcoRII with 1 m
M
AdoHcy and increasing concentrations of
duplex I¢ (1,2,3.5and10n
M
).
Table 3. Binding and substrate properties of 2P-containing DNA duplexes. Apparent dissociation constants (K
app
d
) of complex M.EcoRII–DNA–
AdoHcy were calculated as described in Materials and methods. Relative K

0
(n
M
Æmin
)1
) V
rel
0
(%)
I
5¢-GCCAACCTGGCTCT-3¢/ 4.9 ± 1.8 100 ± 37 195.6 ± 39.1 100 ± 20
3¢-CGGTTGGACCGAGA-5¢
II
5¢-GCCAA
2P
CTGGCTCT-3¢/ 3.9 ± 1.4 80 ± 28 170.2 ± 56.7 87 ± 29
3¢-CGGTT-GGACCGAGA-5¢
III
5¢-GCCAAC
2P
TGGCTCT-3¢/ 5.3 ± 2.4 108 ± 49 3.5 ± 0.3 1.8 ± 0.1
3¢-CGGTTG-GACCGAGA-5¢
IV
5¢-GCCAACC
2P
GGCTCT-3¢/ 96.0 ± 35.6 1959 ± 726 41.1 ± 11.7 21 ± 6
3¢-CGGTTGG-ACCGAGA-5¢
Ó FEBS 2004 2-Pyrimidinone as a probe for DNA methyltransferase (Eur. J. Biochem. 271) 2395
covalent M.EcoRII complex with duplex III can be formed
(see below). Thus, the K

of T by 2P (duplex IV), the pattern of functional groups
exposed into the minor groove remains the same, with the
groups of the central thymine residue exposed into the
major groove being disturbed. Therefore, it is likely that
weak binding of duplex IV to M.EcoRII may be attributed
to the elimination of some DNA–protein contacts in the
major groove of the double helix. Alternatively, this effect
may be caused by a greater destabilization of duplex IV in
comparison with duplexes II and III (Table 1). However,
the conformations of duplexes I–IV are similar. It has also
been shown that substitution of AT by CI in the 5¢…GGT/
ACC…3¢ sequence for SinI C5 MTase led to a considerable
increase in K
m
[13]. This observation corresponds to our
suggestion that specific contacts of C5 MTases with the
central base pair could be mediated by contacts not only in
the minor but also in the major groove.
Comparison of methylation of unmethylated and hemi-
methylated DNA duplexes (Tables 2 and 3) permits us to
speculate about influence of 2P on methylation of unmodi-
fied DNA strand in duplexes II–IV. Equal methylation rates
of duplexes II and IIm allow us to suggest that rates of
methylation of unmodified and 2P-containing strands in
duplex II are virtually the same. Analogously, we suppose
equal methylation rates of unmodified and 2P-containing
strands in duplex IV. The unmodified strand in duplex III
was not methylated under steady-state conditions – prob-
ably due to formation of the stable covalent adduct of
M.EcoRII with 2P-containing strand.

oligonucleotides. These products seem to be oligonucleo-
tides generated from the duplex III–M.EcoRII adducts. The
SDS gel (Laemmli) exhibits two components at different
pH: an upper part at pH 6.8 (stacking gel) and a lower part
at pH 8.8 (separating gel) (Fig. 5B). Due to a pH change
from 6.8 to 8.8, b-elimination of the proton from the C5
position of 2P and dissociation of the covalent intermediates
of M.EcoRII and duplex III take place. The appearance of
the slowly moving oligonucleotides is attributed to retarda-
tion of the duplex III–M.EcoRII covalent intermediates in
the upper part of the gel before dissociation.
It is interesting to compare the stabilities of the adducts of
C5 MTases with DNA duplexes containing AzaC, FC or
2P in place of the target C in the presence of AdoMet. The
adducts of M.EcoRII with AzaC DNA [5] and M.HhaI
with FC DNA [27] are resistant to heating in SDS solution
Fig. 4. Binding of M.EcoRII to DNA duplex I in the presence of
AdoHcy. Relative amount of M.EcoRII–DNA–AdoHcy complex
obtained from the gel-shift autoradiograph vs. protein concentration is
plotted. M.EcoRII (1.5–94 n
M
) was incubated with duplex I (15 n
M
)in
the presence of AdoHcy (1 m
M
). Inset, autoradiograph of gel-shift
assay of M.EcoRII with duplex I. Lanes: 1–8, duplex I with 1 m
M
AdoHcy and increasing concentrations of M.EcoRII (5, 6, 7, 10, 40,

the case of duplex 5¢-GAGCCAAGCGCACTCTGA-3¢/
3¢-CTCGGTTCGCGTGAGACT-5¢(V) lacking the EcoR-
II recognition sequence (Fig. 6). There was also no methy-
lation in a control sample containing the same amount of
enzyme and AdoMet but no DNA. Methylation of duplex
III may be due to methyl transfer to the target unmethylated
cytosine residue. However, this is impossible in the case of
duplex IIIm. Hence, one can suggest that a methyl group
transfer occurs to the 2-pyrimidinone base.
The methylation of duplex IIIm may be stopped at the
stage of formation of the covalent intermediate (Fig. 1B,
step1) or may proceed with dissociation of the covalent
intermediate and release of the methylated 2P-containing
DNA (Fig. 1B, step2). In the first case, the quantity of methyl
groups incorporated into duplex IIIm should correspond to
the quantity of methyl groups incorporated into canonical
DNA after the first turnover of the methylation reaction. In
the second case, we should observe more than one turnover
of the methylation reaction for duplex IIIm. To clarify the
nature of this new effect, we compared the dependence of
methylation of duplexes Im and IIIm on an enzyme
concentration (Fig. 6). Complete methylation of duplex Im
was observed even at low enzyme concentration. M.EcoRII
transfers the methyl group to unmodified DNA strand, turns
Fig. 6. Dependence of methylation of unmethylated (III), hemimethyl-
ated (Im and IIIm) and nonspecific (V) DNA duplexes on concentration of
M.EcoRII. M.EcoRII was incubated with indicated duplexes (500 n
M
)
in buffer B in the presence of [CH

Stacking (upper) and separating (lower) components of the gel are
shown schematically.
Ó FEBS 2004 2-Pyrimidinone as a probe for DNA methyltransferase (Eur. J. Biochem. 271) 2397
over several times and, as a result, methylates all target
cytosine residues for 30 min. The observed level of methy-
lation of duplex IIIm was low. There was a linear increase of
methylation with the increase of the enzyme concentration.
This effect may be due to the arrest of the reaction after one
turnover. One can suggest that the stable covalent adduct
between M.EcoRII and 2P residue in DNA was formed. Its
amount grew with the increase of the enzyme concentration.
Therefore, for duplex IIIm, inhibition of M.EcoRII by 2P-
containing DNA (i.e. the covalent intermediate is formed)
occurs with methyl group transfer to the C5 position of 2P,
and all active enzyme molecules become covalently bound to
2P-containing DNA (Fig. 1B, step 1). In duplex III, only one
strand is modified. However, the level of methylation of
duplex III was unexpectedly low (Fig. 6). We suppose that
formation of the stable covalent adduct with strand
containing 2P prevents effective methylation of the duplex
III unmodified strand.
The time dependence of methyl transfer to duplexes Im
and IIIm was studied (data not shown). Most of the methyl
groups were transferred to duplexes Im and IIIm by
M.EcoRII within 1–2.5 min. During the remainder of the
time there was very little or no further methyl transfer to
DNA. We suggest that duplex IIIm forms a covalent adduct
with M.EcoRII within the first few minutes of the reaction.
Taken together, the results obtained suggest that the
mechanism of C5 MTases inhibition by 2P in the presence

ing DNA in the presence of AdoMet causes the inhibition
of methylation. One can suggest that the potency of
2-pyrimidinone as an inhibitor arises from the retardation
of proton elimination from the covalent intermediate in the
course of catalysis as a consequence of the absence of the N4
amino group in the pyrimidinone ring.
In summary, our data suggest that the conformation of
DNA is not markedly affected by substitution of 2P for
C or T in the sequences studied. 2-Pyrimidinone signifi-
cantly destabilizes the DNA double helix in the order of
sequence contexts: ACCTG > A2PCTG > AC2PTG>
ACC2PG. The amino group of the outer cytosine residue
in the recognition sequence does not take part in the
recognition of DNA by M.EcoRII. Functional groups of
the central thymine exposed in the major groove are
probably involved in the recognition by the enzyme.
EcoRII C5 MTase is inhibited by DNA containing
2-pyrimidinone instead of the target cytosine, two types
of covalent intermediates are possible depending on the
presence of AdoMet or AdoHcy. Both types of adducts
undergo decomposition under heating in the presence of
SDS or under analysis by SDS/PAGE. The revised
mechanism of inhibition of C5 MTases by 2-pyrimidinone
containing DNA may be useful in the application of
2-pyrimidinone containing DNA as a MTase inhibitor.
2-pyrmidinone incorporation in DNA sequences may also
serve as a specific probe for studying discrimination
contacts formed by proteins and functional groups of
pyrimidine bases exposed in the major groove of DNA.
Acknowledgements

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