The stereochemistry of benzo[a]pyrene-2¢-deoxyguanosine
adducts affects DNA methylation by SssI and HhaI DNA
methyltransferases
Oksana M. Subach
1
, Diana V. Maltseva
1
, Anant Shastry
2
, Alexander Kolbanovskiy
2
,
Saulius Klimas
ˇ
auskas
3
, Nicholas E. Geacintov
2
and Elizaveta S. Gromova
1
1 Chemistry Department, Moscow State University, Russia
2 Department of Chemistry, New York University, NY, USA
3 Laboratory of Biological DNA Modification, Institute of Biotechnology, Vilnius, Lithuania
The polycyclic aromatic hydrocarbons are a well-
known class of ubiquitous environmental pollutants
which are generated by incomplete combustion of
organic matter. These compounds require metabolic
activation to highly reactive diol epoxides to elicit their
detrimental genotoxic effects [1]. Benzo[a]pyrene
(B[a]P), one of the most widely studied polycyclic
aromatic hydrocarbons, is metabolically activated

2
-dG and (+)-trans-B[a]P-N
2
-dG adducts in complexes with
MTases are enhanced, but to different extents, indicating that aromatic
B[a]P residues are positioned in different microenvironments in the DNA–
protein complexes. We have previously shown that the (+)-trans-isomeric
adduct inhibits both the binding and methylating efficiencies (k
cat
) of both
MTases [Subach OM, Baskunov VB, Darii MV, Maltseva DV, Alexandrov
DA, Kirsanova OV, Kolbanovskiy A, Kolbanovskiy M, Johnson F,
Bonala R, et al. (2006) Biochemistry 45, 6142–6159]. Here we show that the
stereoisomeric (+)-cis-B[a]P-N
2
-dG lesion has only a minimal effect on the
binding of these MTases and on k
cat
. The minor-groove (+)-trans adduct
interferes with the formation of the normal DNA minor-groove contacts
with the catalytic loop of the MTases. However, the intercalated base-
displaced (+)-cis adduct does not interfere with the minor-groove DNA–
catalytic loop contacts, allowing near-normal binding of the MTases and
undiminished k
cat
values.
Abbreviations
AdoHcy, S-adenosyl-
L-homocysteine; AdoMet, S-adenosyl-L-methionine; B[a]P, benzo[a]pyrene; B[a]PDE, r7,t8-dihydroxy-t9,10-epoxy-
7,8,9,10-tetrahydrobenzo[a]pyrene; B[a]P-DNA, DNA containing benzo[a]pyrene; C5 MTase, C5-cytosine DNA methyltransferase; EMSA,

enantiomer (–)-7S,8R-diol 9R,10S diol epoxide of
B[a]P is not formed in eukaryotic cells [8], it is often
used in structure–function studies of B[a]P-N
2
-dG
adducts because of the different conformational char-
acteristics of the (–)-trans-B[a]P-N
2
-dG and (+)-trans-
B[a]P-N
2
-dG adducts [6].
The structures of (+)-trans-B[a]P-N
2
-dG and (+)-cis-
B[a]P-N
2
-dG adducts in dsDNA are very different
from one another, the former being characterized by
an external minor-groove conformation and the latter
by a base-displaced intercalative conformation [6,9,10].
The different structural characteristics have a pro-
nounced effect on the cellular processing of these stereo-
isomeric DNA adducts. First, both prokaryotic and
eukaryotic nucleotide excision repair systems eliminate
the (+)-cis-B[a]P-N
2
-dG adducts more efficiently than
the (+)-trans-B[a]P-N
2

[20]. The greatest disturbance of DNA cleavage is
caused by the (+)-trans-B[a]P-N
2
-dG and (+)-cis-
B[a]P-N
2
-dG adducts [20,21]. In the present work, we
explored the hypothesis that DNA methylation is
dependent on the absolute configurations and confor-
mations of (+)-trans-B[a]P-N
2
-dG and (+)-cis-B[a]P-
N
2
-dG lesions.
DNA methylation plays an important role in dif-
ferent cellular processes such as regulation of tran-
scription, cell development, and chromatin structure
[22,23]. Mammalian genomes are methylated at cer-
tain CpG sites, resulting in different patterns of
DNA methylation [22,23]. Disruption of methylation
patterns can lead to cancer [24–28]. In eukaryotes,
methylation of CpG sites is carried out by several
C5-cytosine DNA methyltransferases (C5 MTases;
EC 2.1.1.37). Prokaryotic C5 MTases are good mod-
els of biological methylation because they share with
mammalian C5 MTases a number of conserved
amino-acid motifs that have structural roles and are
involved in catalysis [29]. The prokaryotic C5 MTases
SssI and HhaI transfer a methyl group to the C5

-dG
Y
+
- (+)-trans-B[a]P-N
2
-dG
Fig. 1. Chemical structures of the (+)-trans-B[a]P-N
2
-dG and (+)-cis-
B[a]P-N
2
-dG adducts.
Stereochemistry of B[a]P-N
2
-dG affects methylation O. M. Subach et al.
2122 FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS
B[a]P-N
2
-dG lesions are formed efficiently at the
guanine residue in CpG sequence contexts [31] that are
recognition sites of mammalian MTases. The efficiency
of such damage is enhanced in the presence of m
5
dC
instead of dC in 5¢-CpG targets [31–33]. Such damage
in the promoter region of a gene may disturb the nor-
mal functioning of MTases and change the genomic
methylation pattern. It has previously been found that
the concentrations of methylated cytosines in the
DNA of mammalian cells treated with racemic

2
-dG adduct conforma-
tions, it is of structural interest to compare the effects
of these conformations on DNA methylation. In this
work, the effect of the intercalated [9] (+)-cis-anti-
B[a]P-N
2
-dG adduct on the DNA binding and catalytic
activity of SssI and HhaI was examined and compared
with the effects of the minor-groove (+)-trans-B[a]
P-N
2
-dG adduct [37]. The hypothesis was tested that
the (+)-cis-B[a]P-N
2
-dG adducts, because of their
intercalative conformations, inhibit methylation to a
lesser extent because the DNA minor groove remains
available for interaction with the critical amino-acid
groups of the MTases. Using biochemical and spectro-
scopic methods, we show here that the (+)-cis-anti-
B[a]P-N
2
-dG adducts indeed do not significantly
inhibit methylation, demonstrating that the stereo-
chemistry of B[a]P metabolite-derived DNA adducts
can affect this potentially important epigenetic mech-
anism of cancer initiation [1,38].
Results
The (+)-cis-B[a]P-N

GCG 5¢-CACCCTTGCGCTCTCTCA
CGC 5¢-TGAGAGAGCGCAAGGGTG
CGM 5¢-TGAGAGAGMGCAAGGGTG
X
+
CG 5¢-CACCCTTX
+
CGCTCTCTCA
GCX
+
5¢-CACCCTTGCX
+
CTCTCTCA
Y
+
CG 5¢-GAGCCAAY
+
CGCACTCTGA
GCY
+
5¢-GAGCCAAGCY
+
CACTCTGA
Table 2. Properties of the oligodeoxynucleotide duplexes containing (+)-cis-B[a]P-N
2
-dG adduct as substrates of M.HhaI and M.SssI. The tar-
get dC are underlined. M.SssI ⁄ M.HhaI sites are in bold. The other designations are as in Table 1.
Designation DNA duplex
M.HhaI M.SssI
K

CTCTCTCA
3¢-GTGGGAA
CGCGAGAGAGT
42.8 ± 13.9 2.0 ± 0.2 3.8 ± 0.6 0.7 ± 0.2
G
CG ⁄ CGM 5¢-CACCCTTGCGCTCTCTCA
3¢-GTGGGAA
CGMGAGAGAGT
13.0 ± 3.9 2.1 ± 0.3 4.1 ± 0.8 0.4 ± 0.2
X
+
CG ⁄ CGM 5¢-CACCCTTX
+
CGCTCTCTCA
3¢-GTGGGAA
C GMGAGAGAGT
61 ± 14 1.5 ± 0.3 4.2 ± 0.5 0.13 ± 0.05
G
CX
+
⁄ CGM 5¢-CACCCTTGCX
+
CTCTCTCA
3¢-GTGGGAA
CGM GAGAGAGT
13.6 ± 2.5 1.7 ± 0.2 1.9 ± 0.2 0.5 ± 0.1
O. M. Subach et al. Stereochemistry of B[a]P-N
2
-dG affects methylation
FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2123

and (+)-cis-B[a]P-N
2
-dG adducts
To examine how the stereochemical and conforma-
tional features of the (+)-cis-B[a]P-N
2
-dG (X
+
) and
(+)-trans-B[a]P-N
2
-dG (Y
+
) adducts are affected by
the binding of the MTases, the fluorescence properties
of the pyrenyl residues were examined when the oligode-
oxynucleotide duplexes were titrated with various
amounts of M.HhaI or M.SssI. The duplexes containing
Y
+
are defined in Table 3. The Y
+
residues were intro-
duced into the overlapping recognition sites of both
M.SssI (
CpG) and M.HhaI (GCGC) on either the 5¢-side
(Y
+
CG ⁄ CGM) or the 3¢-side (GCY
+

-dG
adduct in the HhaI recognition site results in a similar
3.5-fold and fourfold increase in the fluorescence
Table 3. Oligodeoxynucleotide duplexes containing (+)-trans-B[a]
P-N
2
-dG adduct. N is any nucleotide residue. The other designa-
tions are as in Tables 1 and 2.
Designation DNA duplex
G
CG ⁄ CGM 5¢-GAGCCAAGCGCACTCTGA
3¢-CTCGGTT
CGMGTGAGACT
Y
+
CG ⁄ CGM 5¢-GAGCCAAY
+
CGCACTCTGA
3¢-CTCGGTT
C GMGTGAGACT
G
CY
+
⁄ CGM 5¢-GAGCCAAGCY
+
CACTCTGA
3¢-CTCGGTT
CGM GTGAGACT
Y
+

+
⁄ CGM
(s), G
CY
+
⁄ CGM (r), and Y
+
CG ⁄ CGM (j), or 200 nM of X
+
CG ⁄
CGM (n) were titrated with M.HhaI in buffer D at 25 °C and then
the emission at 384 nm was measured with excitation at 350 nm.
(C) 100 n
M GCX
+
⁄ CGM (s), X
+
CG ⁄ CGM (n), Y
+
CG ⁄ CGM (j),
G
CY
+
⁄ CGM (r)orY
+
(N)
4
CG ⁄ C(N)
4
GM (·) were titrated with

dG adduct in the recognition site or in the flanking
sequence leads to a 1.6–1.7-fold increase in the fluores-
cence emission intensity of the B[a]P residue (Fig. 2C).
The fluorescence intensity of the B[a]P residue increa-
ses by factors of 2.8–16 upon the binding of M.SssI to
the Y
+
CG ⁄ CGM, GCY
+
⁄ CGM and Y
+
(N)
4
CG ⁄
C(N)
4
GM duplexes containing (+)-trans-B[a]P-N
2
-dG
adduct. Thus, the fluorescence enhancement depends
on the position of the (+)-trans adduct. When M.SssI
binds to G
CY
+
⁄ CGM duplexes containing (+)-trans-
B[a]P-N
2
-dG adducts in the recognition site, the fluor-
escence intensity increases by a factor of 16. Upon
M.SssI binding to Y

The binding of M.SssI and M.HhaI to the oligodeoxy-
nucleotide duplexes was performed in the presence of
the cofactor analog S -adenosyl- l-homocysteine (Ado-
Hcy). In the case of C5 MTases, AdoHcy facilitates
the formation of specific complexes with DNA [41,42].
To determine the K
d
values of the M.SssI or M.HhaI
complexes with the oligodeoxynucleotide duplexes con-
taining the (+)-cis-B[a]P-N
2
-dG adduct, we used a
competition equilibrium binding assay. In these com-
petition experiments, unlabeled B[a]PDE-modified and
32
P-labeled GCG ⁄ CGM
ref
duplexes were mixed before
the addition of MTase. The formation of the com-
plexes of M.SssI and M.HhaI with DNA was moni-
tored by electrophoretic mobility shift assay (EMSA)
(Fig. 3A,B). The competition curves (Fig. 3C,D) are
characteristic of equilibrium competition processes
[43].
In the case of M.HhaI, the K
d
values for the
B[a]PDE-modified X
+
CG ⁄ CGC and GCX

CG ⁄ CGM•AdoHcy and M.SssI•GCX
+
⁄
CGM•AdoHcy complexes, the K
d
values are about the
same as the K
d
of the M.SssI•GCG ⁄ CGM•AdoHcy
complex. Thus, for both enzymes, the K
d
values of
the ternary MTase•(unmethylated cis-B[a]P-DNA)•
AdoHcy and MTase•(hemimethylated cis-B[a]P-DNA)•
AdoHcy complexes are comparable to the K
d
values of
the ternary complexes of MTases with the correspond-
ing unmodified unmethylated (G
CG ⁄ CGC) or hemi-
methylated (G
CG ⁄ CGM) duplexes.
Steady-state kinetics of methylation of
oligodeoxynucleotide duplexes containing
(+)-cis-B[a]P-N
2
-dG adduct by M.SssI and M.HhaI
The rates of methylation of the X
+
CG ⁄ CGC, GCX

cat
values of the corresponding unmodified duplexes.
In the case of M.SssI, the largest effect on DNA
methylation was a 3.1-fold decrease in k
cat
for the
hemimethylated X
+
CG ⁄ CGM duplex containing (+)-
cis-B[a]P-N
2
-dG on the 5¢-side of the target dC residue.
The k
cat
value for the hemimethylated duplex
G
CX
+
⁄ CGM was about the same as that for the
G
CG ⁄ CGM duplex. The k
cat
values determined for
the unmethylated X
+
CG ⁄ CGC and GCX
+
⁄ CGC
duplexes were only 1.3 times smaller than k
cat

matic ring system in the (+)-trans-B[a]P-N
2
-dG
adducts is positioned in the minor groove and is 5¢-
directed relative to the modified guanine residue with
all base pairs intact, including the modified G*•C base
pair [10]. In contrast, the (+)- cis-B[a]P-N
2
-dG adduct
assumes an intercalated base-displaced adduct confor-
mation with the modified dG residue and the partner
base dC in the opposite strand displaced into the
minor and major grooves, respectively [9]. Molecular
views of these (+)-cis and (+)-trans adduct conforma-
tions are shown in Fig. 5A.
The structure of the MTase–DNA complexes con-
taining B[a]P-N
2
-dG adducts in the MTase recognition
sites has not been studied. According to the available
crystal structures of complexes of M.HhaI with
unmodified DNA and AdoHcy(AdoMet) [44], M.HhaI
consists of two domains, the large domain containing
the S-adenosyl-l-methionine (AdoMet) binding site
and the catalytic center, and the small domain contain-
ing the target recognition domain (Fig. 5B). The DNA
molecule is located in the cleft formed between the two
domains with the major groove facing the small
domain and the minor groove facing the large domain.
Before methylation, the target dC residue flips out of

itor X
+
CG ⁄ CGM were 0, 0.5, 1, 5, 10, 40,
80, 120, 200 n
M in lanes 1–9, respectively
(A), and 0, 10, 20, 50, 100, 200, 300, 400,
500 n
M in lanes 1–9, respectively (B). Equi-
librium competition curves for complexes of
M.HhaI (C) and M.SssI (D) with
32
P-labeled
duplex GCG ⁄ CGM
ref
in the presence of
increasing concentrations of the competitor
duplexes G
CG ⁄ CGC(j), X
+
CG ⁄ CGC(m),
G
CX
+
⁄ CGC(d), GCG ⁄ CGM (h),
X
+
CG ⁄ CGM (n)orGCX
+
⁄ CGM (s). The
relative fraction of bound

recent modeling study [46].
Fluorescence properties
The fluorescence of the B[a]P residues is quenched
by factors of 100–200 in (+)-trans-B[a]P-N
2
-dG and
(+)-cis-B[a]P-N
2
-dG mononucleoside adducts [47] by
a solvent-dependent proton-coupled electron-transfer
mechanism [48]. The fluorescence lifetimes are 1.4 ±
0.1 and 0.71 ± 0.2 ns, respectively, in aqueous solu-
tions [47], but are longer in oligonucleotide duplexes.
For example, the fluorescence decay profiles of the
(+)-cis-B[a]P-N
2
-dG and (+)-trans-B[a]P-N
2
-dG with-
in oligonucleotide duplexes are well described by the
sums of three exponential decay components with
mean lifetimes of 4.0 ± 0.2 and 2.4 ± 0.2 ns
(Y. Tang, A. Durandin, and N. E. Geacintov, unpub-
lished). Thus, in the absence of protein, the fluores-
cence characteristics of the (+)-trans-B[a]P-N
2
-dG and
(+)-cis-B[a]P-N
2
-dG adducts are not too different, a

B[a]P-N
2
-dG adduct and a 2.8–16-fold upon binding
of M.SssI to the duplexes containing (+)-trans-B[a]P-
N
2
-dG adduct. Therefore, the larger enhancement of
the fluorescence yield of the (+)-trans adduct relative
to the (+)-cis adduct reflects the difference in the local
microenvironments of the two aromatic pyrenyl resi-
dues in the protein–DNA complexes.
It is known from previous studies that the fluores-
cence yields of (+)-trans-B[a]P-N
2
-dG mononucleoside
adducts are dramatically increased as the concentration
of organic solvents is increased in aqueous mixtures
[53]. The differences in the fluorescence yields upon
formation of the M.HhaI•G
CY
+
⁄ CGM•AdoHcy
and M.HhaI•Y
+
CG ⁄ CGM•AdoHcy complexes sug-
gest that the (+)-trans adducts are situated in a
more hydrophobic environment in the protein com-
plexes than in aqueous solution in the absence of
A
B

thesis is that the change in the hydrophobicity of the
local environment upon protein binding is less pro-
nounced in the case of the (+)-cis-B[a]P-N
2
-dG adduct
than in the case of the (+)-trans-B[a]P-N
2
-dG adduct.
Thus, the different microenvironment of the pyrenyl
residue in the (+)-cis-B[a]P-N
2
-dG adduct and (+)-
trans-B[a]P-N
2
-dG adduct in MTase•B[a]P-DNA com-
plexes is revealed by fluorescence studies.
It has been postulated that the flipping or extrusion
of the target base from the DNA duplex is an import-
ant intermediate step in DNA methylation catalyzed
by C5 MTases [54]. We postulated that the fluores-
cence of the pyrenyl residue in the B[a ]P-N
2
-dG
adducts would be particularly sensitive to changes in
the microenvironment when this adduct is flanked by a
target cytosine that undergoes flipping in the MTase–
DNA complexes. In accordance with this, the depend-
ence of the fluorescence of the (+)-trans adduct on its
position relative to the target dC was revealed in the
case of the formation of the complexes of M.SssI

(N)
4
CG ⁄ C(N)
4
GM dup-
lex, the fluorescence enhancement upon formation
of the M.SssI–DNA complex is significantly smaller
(Fig. 2C). In the case of the Y
+
CG ⁄ CGM duplex,
when the B[a]P aromatic ring system is out of the
CpG site but near the target dC, the fluorescence
A
B
Fig. 5. (A) Conformations of the B[a]PDE-
modified duplexes containing the (+)-trans-
B[a]P-N
2
-dG and (+)-cis-B[a]P-N
2
-dG adducts
obtained by NMR methods and adapted
from [62] with permission of the American
Chemical Society. (B) Three-dimensional
structure of the ternary complex of M.HhaI
with the 12-mer duplex containing GCGC
and the cofactor analog AdoHcy derived
from the RCSB Protein Data Bank (3mht
[63]). The catalytic loop, the flipped out cyto-
sine, and AdoHcy are depicted in dark grey.

-dG and
(+)-trans-B[a]P-N
2
-dG adducts in different sequence
contexts are compared with one another in Fig. 6. The
minor-groove position of the (+)-trans-B[a]P-N
2
-dG
adduct did not significantly affect M.SssI binding to
DNA, but reduced M.HhaI binding by 1–2 orders of
magnitude (Fig. 6). Therefore, the bulky B[a]P residue
positioned in the DNA minor groove severely inhibits
DNA binding to M.HhaI by perturbing the minor-
groove DNA–M.HhaI contacts and does not signifi-
cantly influence DNA binding to M.SssI [37]. Our
observations indicate that the introduction of the (+)-
cis-B[a]P-N
2
-dG into DNA does not cause any signifi-
cant changes in K
d
for either M.SssI or M.HhaI
(Table 2, Fig. 6). This observation can be accounted
for by the intercalative conformation of the B[a]P resi-
dues in the (+)-cis adducts which interferes less signifi-
cantly with DNA–protein interactions on either side of
the modified base pair. Thus, the stereochemistry of
the B[a]P-N
2
-dG adducts in DNA does not influence

with the interactions between the catalytic loops of
SssI and HhaI MTases and the minor groove of the ol-
igodeoxynucleotide duplexes [37]. However, in the case
of the (+)-cis-B[a]P-N
2
-dG adduct in the unbound
duplex, the B[a]P residue is intercalated into the DNA
A
B
Fig. 6. Bar graphs representing relative K
d
(K
rel
d
) and k
cat
(k
rel
cat
) values for binding and
methylation of DNA containing (+)-cis-B[a]P-
N
2
-dG and (+)-trans-B[a]P-N
2
-dG adducts by
M.SssI (A) and M.HhaI (B). The K
rel
d
and k

). The target dC residue is underlined.
O. M. Subach et al. Stereochemistry of B[a]P-N
2
-dG affects methylation
FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2129
helix, and the modified dG residue is displaced into
the minor groove. These findings suggest that the
B[a]P aromatic ring system remains stacked between
neighboring base pairs, thus exerting relatively minor
effects on K
d
and k
cat
. In this model, the bulky B[a]P
residue does not significantly disturb the contacts
between the M.HhaI (or M.SssI) catalytic loops
with the minor groove of the oligodeoxynucleotide
duplexes.
Relative to the unmodified G
CG ⁄ CGM duplex, a
small decrease in the efficiency of methylation by
M.SssI of the X
+
CG ⁄ CGM duplex is observed when
the (+)-cis adduct X
+
is positioned on the 5¢-side of
the target dC residue (Fig. 6, Table 2). On the other
hand, k
cat

dG or (+)-trans-B[a]P-N
2
-dG adduct in complexes
with MTases are enhanced, but to different extents,
indicating that aromatic B[a]P residues are positioned
in different microenvironments in these DNA–protein
complexes. Such effects of adduct stereochemistry on
hypomethylation may also exist in the case of mamma-
lian MTases, and these possibilities are being investi-
gated in our laboratory.
Experimental procedures
Chemicals and enzymes
AdoMet and AdoHcy were purchased from Sigma (St
Louis, MO, USA). [CH
3
-
3
H]AdoMet (77 CiÆmmol
)1
,
13 lm) was from Amersham Biosciences (Little Chalfont,
UK). [c-
32
P]ATP (1000 CiÆmmol
)1
) was bought from Izotop
(Obninsk, Russia). M.HhaI (4.4 mgÆmL
)1
) was prepared as
described previously [57]. Also we used His

Oligodeoxynucleotides
The sequences of the oligodeoxynucleotides used are sum-
marized in Table 1. GCG
ref
, CGM
ref
, GCG, CGC and
CGM were purchased from IDT (Coralville, IA, USA) and
Syntol (Moscow, Russia).
Y
+
CG and GCY
+
oligodeoxynucleotides containing a
single (+)-trans-B[a]P-N
2
-dG adduct were obtained as des-
cribed [49]. The site-specifically modified X
+
CG and
GCX
+
oligodeoxynucleotides containing a single (+)-cis-
B[a]P-N
2
-dG lesion were obtained by treatment of GCG
with racemic B[a]PDE solution using previously described
methods [59]. The (+)-trans-B[a]P-N
2
-dG, (–)-trans-B[a]

CG ⁄ CGM, GCY
+
⁄ CGM and Y
+
(N)
4
CG ⁄ C (N)
4
GM
duplexes was recorded on a Perkin–Elmer spectrofluorime-
ter with slit widths of 5–10 nm for excitation and 3–5 nm
for the emission monochromator. All titrations were per-
formed in a micro quartz cuvette (10 mm · 10 mm,
100 lL; Starna Cells, Atascadero, CA, USA). X
+
CG ⁄
Stereochemistry of B[a]P-N
2
-dG affects methylation O. M. Subach et al.
2130 FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS
CGM, GCX
+
⁄ CGM, Y
+
CG ⁄ CGM, G CY
+
⁄ CGM or
Y
+
(N)

CG
C, GCX
+
⁄ CGC, X
+
CG ⁄ CGM and GCX
+
⁄ CGM, the
unmodified strands CGC or CGM were mixed with a two-
fold excess of the B[a]PDE-modified strands, X
+
CG or
GCX
+
, in buffer A, or in 50 mm Tris•HCl (pH 7.5) ⁄
50 mm NaCl. The mixtures of oligodeoxynucleotides were
heated to 80 °C and allowed to cool to room temperature.
The MTases HhaI and SssI do not bind X
+
CG or GCX
+
strands (data not shown). To obtain unmodified GCG ⁄
CG
C and GCG ⁄ CGM duplexes, the oligodeoxynucleotide
strands were mixed in the ratio 1 : 1. In the case of M.SssI,
the reference
32
P-labeled GCG ⁄ CGM
ref
duplex (100 nm)

ref
•AdoHcy and MTase•B[a]P-DNA•
AdoHcy complexes. The k ratio specifies the relative bind-
ing efficiency of the reference unmodified DNA and the
competitor damaged DNA to the MTase. The K
d
values
were calculated by dividing K
r
d
(see below) by the experi-
mental values of k.
Determination of K
r
d
for the M.HhaI(M.SssI)•
G
CG ⁄ CGM
ref
•AdoHcy complexes
We were unable to obtain an accurate K
r
d
values for the
M.HhaI•G
CG ⁄ CGM
ref
•AdoHcy complex by direct titra-
tion of solutions of the G
CG ⁄ CGM

Hcy complex was obtained by direct titration using EMSA.
The
32
P-labeled oligodeoxynucleotide duplex, Y
+
CG ⁄ CGM
(0.2 nm), was incubated in the presence of 0.1 mm AdoHcy
with various M.HhaI concentrations (0.3–5 nm) in buffer E
containing 8% glycerol at 37 °C for 5 min and at 0 °C for
10 min. The further experimental procedures and data ana-
lysis were the same as described in [37]. The K
r
d
value for
the M.SssI•G
CG ⁄ CGM
ref
•AdoHcy complex was deter-
mined as described [37].
Methylation assay
Oligodeoxynucleotide duplexes were obtained as described
above. The B[a]PDE-modified X
+
CG or GCX
+
strands
alone are not methylated by MTases HhaI and SssI (data
not shown). The efficiency of methylation was monitored
by the radioactivity of tritium (CH
3

tion mixtures were pipetted on to DE-81 paper disks (What-
man, Brentford, UK) and treated as described [61]. The
amounts of methylated DNA were computed as described
[60]. The V
0
values for all duplexes were determined from the
initial linear portions of the product versus time profiles. In
O. M. Subach et al. Stereochemistry of B[a]P-N
2
-dG affects methylation
FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2131
the used duplex concentration ranges, the measured V
0
values were constant for each unmodified or B[a]PDE-modi-
fied duplex, indicating that the V
max
limit was reached. Using
the V
max
values thus obtained, the k
cat
values were calculated
(Table 2).
Acknowledgements
This research was supported by a US Public Health
Service grant No. TW05689 from the Fogarty Interna-
tional Center (New York University and Moscow
State University), NIH Grant CA 099194 (New York
University), and RFBR Grants 04-04-49488 and 05-04-
49690 (Moscow State University). We thank Dr

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