The fluorescence properties of the pyrenyl residues of the +-cis-B[a]P-N2-dG and +-trans-B[a]P-N2-dG adducts in complexes with MTases are enhanced, but to different extents, indicating th
Trang 1adducts affects DNA methylation by SssI and HhaI DNA methyltransferases
Oksana M Subach1, Diana V Maltseva1, Anant Shastry2, Alexander Kolbanovskiy2,
Saulius Klimasˇauskas3, Nicholas E Geacintov2and Elizaveta S Gromova1
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
in vivo to the highly mutagenic [2] and tumorigenic [3] (+)-7R,8S-diol 9S,10R-epoxide of benzo[a]pyrene
Keywords
benzo[a]pyrene-2¢-deoxyguanosine adducts;
DNA methyltansferases; environmental
pollutants; stereochemistry
Correspondence
E S Gromova, Chemistry Department,
Moscow State University, Moscow,
119992, Russia
Fax: +7495 939 31 81
Tel: +7495 939 31 44
E-mail: gromova@genebee.msu.ru
(Received 19 July 2006, revised 19 January
2007, accepted 21 February 2007)
doi:10.1111/j.1742-4658.2007.05754.x
The biologically most significant genotoxic metabolite of the environmental pollutant benzo[a]pyrene (B[a]P), (+)-7R,8S-diol 9S,10R-epoxide, reacts chemically with guanine in DNA, resulting in the predominant formation
of (+)-trans-B[a]P-N2-dG and, to a lesser extent, (+)-cis-B[a]P-N2-dG adducts Here, we compare the effects of the adduct stereochemistry and conformation on the methylation of cytosine catalyzed by two purified prokaryotic DNA methyltransferases (MTases), SssI and HhaI, with the lesions positioned within or adjacent to their CG and GCGC recognition sites, respectively The fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N2-dG and (+)-trans-B[a]P-N2-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 (kcat) 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-N2-dG lesion has only a minimal effect on the binding of these MTases and on kcat 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 kcatvalues
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, electrophoretic mobility shift assay; k cat , multiple turnover rate constant; K d , dissociation constant; M.SssI, SssI DNA methyltransferase; M.HhaI, HhaI DNA methyltransferase; MTase, DNA methyltransferase; V0, initial rate of methylation; Vmax, maximal rate of methylation.
Trang 2{(+)-B[a]PDE} [1] This metabolite reacts with DNA
predominantly at the N2-exocyclic amino groups of
guanine [4,5] via trans or cis opening of the epoxide
ring to form the (+)-trans-B[a]P-N2-dG and
(+)-cis-B[a]P-N2-dG adducts (Fig 1) The relative yields of
the two stereoisomeric adducts are generally higher in
the case of the (+)-trans-B[a]P-N2-dG adduct (in some
cases more than 90%) than in the case of the
(+)-cis-B[a]P-N2-dG adduct (up to 13%) [4,6,7] Although the
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-N2-dG
adducts because of the different conformational
char-acteristics of the (–)-trans-B[a]P-N2-dG and
(+)-trans-B[a]P-N2-dG adducts [6]
The structures of (+)-trans-B[a]P-N2-dG and
(+)-cis-B[a]P-N2-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-N2-dG adducts more efficiently than the (+)-trans-B[a]P-N2-dG adducts [11,12] The lesions that escape repair can influence DNA replication, tran-scription, and the interaction of different proteins with DNA All four B[a]P-N2-dG adducts inhibit DNA repli-cation [13,14] The successful, although error-prone, translesional synthesis past both stereoisomeric adducts has been reported [13,15,16] The fidelity of trans-lesional synthesis depends on adduct stereochemistry, nucleotide sequence context, and the DNA polymerase [15,16] In the case of DNA transcription, T7 RNA polymerase is blocked more efficiently by the (+)-trans-B[a]P-N2-dG adduct than by the (+)-cis-B[a]P-N2-dG adduct [17] The binding of the transcription factor Sp 1
to B[a]PDE-modified DNA is highly dependent on the B[a]P-N2-dG conformation [18], whereas no apparent differences in the binding affinities of the Ap 1 tran-scription factor to DNA containing different stereoiso-meric B[a]P-N2-dG adducts was observed [19] The B[a]P-DNA adducts also affect the function of human topoisomerase I by alteration of DNA cleavage patterns [20] The greatest disturbance of DNA cleavage is caused by the (+)-trans-B[a]P-N2-dG and (+)-cis-B[a]P-N2-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-N2-dG and
(+)-cis-B[a]P-N2-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 position of the target cytosine (C) in their CG and GCGC recognition sites, respectively The M.SssI has substrate specificity identical with that of the mam-malian MTases [30]
HO
HO
OH
N NH O
N
N
R
NH
HO
HO
OH
N
NH O
N
N
R
NH
X+ - (+)-cis-B[a]P-N2-dG
Y+ - (+)-trans-B[a]P-N2-dG
Fig 1 Chemical structures of the (+)-trans-B[a]P-N 2 -dG and
(+)-cis-B[a]P-N2-dG adducts.
Trang 3B[a]P-N2-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 m5dC
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
(+⁄ –)-B[a]PDE are lower than normal [34,35] In these
earlier investigations, only the overall concentrations
of B[a]P-N2-dG adducts were compared with the levels
of DNA methylation, and thus the effect of the
abso-lute configurations and conformations of the adducts
on DNA methylation was not evaluated More
recently, the effect of stereochemically distinct
(+)-trans-B[a]P-N2-dG and (–)-trans-B[a]P-N2-dG adducts
on DNA methylation by the prokaryotic MTases
Eco-RII [36], SssI, and HhaI [37] was examined In most
cases, the methylation efficiency of
oligodeoxynucleo-tide duplexes containing trans adducts in MTase
recog-nition sites by these MTases was diminished These
effects were attributed to the conformation of the
trans-B[a]P-N2-dG adducts in the minor groove of
B-DNA [10], which interfere with the formation of the
normal and critical minor-groove MTase-DNA
con-tacts [37]
Because of the markedly different
(+)-trans-B[a]P-N2-dG and (+)-cis-B[a]P-N2-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-N2-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-N2-dG adduct [37] The hypothesis was tested that the (+)-cis-B[a]P-N2-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-N2-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-N2-dG lesions (X+) were site-specifi-cally incorporated into the single-stranded oligonucleo-tides shown in Table 1 The corresponding duplexes are shown in Table 2 The X+ residues were intro-duced into the overlapping recognition sites of both M.SssI (CpG) and M.HhaI (GCGC) on either the
Table 1 Oligodeoxynucleotide sequences synthesized M, m 5 dC;
X+, (+)-cis-B[a]P-N2-dG; Y+, (+)-trans-B[a]P-N2-dG.
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
Kd(p M ) kcat(min)1) Kd(n M ) kcat(min)1)
3¢-GTGGGAACGCGAGAGAGT
X + CG ⁄ CGC 5¢-CACCCTTX+CGCTCTCTCA
3¢-GTGGGAAC GCGAGAGAGT
GCX+⁄ CGC 5¢-CACCCTTGCX+CTCTCTCA
3¢-GTGGGAACGC GAGAGAGT
3¢-GTGGGAACGMGAGAGAGT
X + CG ⁄ CGM 5¢-CACCCTTX+CGCTCTCTCA
3¢-GTGGGAAC GMGAGAGAGT
GCX + ⁄ CGM 5¢-CACCCTTGCX+CTCTCTCA
3¢-GTGGGAACGM GAGAGAGT
Trang 45¢- or the 3¢-side of the target dC residue In
hemi-methylated oligonucleotide duplexes (X+CG⁄ CGM
and GCX+⁄ CGM), 5-methylcytosine was introduced
into one of the strands of the recognition site instead
of the target cytosine The melting curves of the
hemi-methylated X+CG⁄ CGM and GCX+⁄ CGM duplexes
containing (+)-cis-B[a]P-N2-dG were cooperative with
the melting temperature ranging from 57 to 61C,
being 4–8C lower than that of the unmodified
GCG⁄ CGM duplex (data not shown) Therefore, the
(+)-cis-B[a]P-N2-dG lesions destabilize the 18-mer
duplexes
Effects of M.HhaI and M.SssI binding on the
fluorescence of the (+)-trans-B[a]P-N2-dG
and (+)-cis-B[a]P-N2-dG adducts
To examine how the stereochemical and
conforma-tional features of the (+)-cis-B[a]P-N2-dG (X+) and
(+)-trans-B[a]P-N2-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+⁄ CGM) of the
target dC residue or distant from it [Y+(N)4CG⁄
C(N)4GM] The emission spectra of X+CG⁄ CGM,
GCX+⁄ CGM, Y+CG⁄ CGM and GCY+⁄ CGM
dup-lexes alone or in compdup-lexes with MTases exhibit the
usual broad maxima at 384 and 404 nm (Fig 2A),
con-sistent with those previously reported [39,40]
The binding of M.HhaI to the X+CG⁄ CGM and
GCX+⁄ CGM duplexes containing (+)-cis-B[a]P-N2-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.
3¢-CTCGGTTCGMGTGAGACT
3¢-CTCGGTTC GMGTGAGACT
3¢-CTCGGTTCGM GTGAGACT
Y + (N)4CG ⁄ C(N) 4 GM 5¢-GCTY + GTGGCGTAGGC
3¢-CGAC CACCGMATCCG
Fig 2 Fluorescence titration of DNA containing (+)-cis-B[a]P-N 2 -dG (X+) or (+)-trans-B[a]P-N2-dG (Y+) adducts with M.HhaI or M.SssI (A) Typical fluorescence emission spectra of the M.HhaI•B[a]P-DNA•AdoHcy complexes and the free B[a]P-DNA duplexes; 500 n M GCX+⁄ CGM or GCY + ⁄ CGM duplexes were incubated with 875 n M M.HhaI in the presence of 0.1 m M AdoHcy in buffer D The fluores-cence excitation wavelength was 350 nm (B) 500 n M GCX + ⁄ CGM (s), GCY + ⁄ CGM (r), and Y + CG ⁄ CGM (j), or 200 n M 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), GCY+⁄ CGM (r) or Y +
(N) 4 CG ⁄ C(N) 4 GM (·) were titrated with M.SssI in buffer B at 25 C The excitation and emission wave-lengths are the same as in (B).
Trang 5emission intensity of the aromatic pyrenyl residue,
respectively (Fig 2B) However, in the case of the
(+)-trans-B[a]P-N2-dG adducts, the increase in
fluores-cence intensity is much more pronounced upon the
binding of M.HhaI to the Y+CG⁄ CGM and
GCY+⁄ CGM duplexes (by factors of 30 and 20,
respectively) The fluorescence enhancement in practice
does not depend on the position of the (+)-trans or
(+)-cis adduct
The binding of M.SssI to the X+CG⁄ CGM and
GCX+⁄ CGM duplexes containing (+)-cis-B[a]P-N2
-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)4CG⁄
C(N)4GM duplexes containing (+)-trans-B[a]P-N2-dG
adduct Thus, the fluorescence enhancement depends
on the position of the (+)-trans adduct When M.SssI
binds to GCY+⁄ CGM duplexes containing
(+)-trans-B[a]P-N2-dG adducts in the recognition site, the
fluor-escence intensity increases by a factor of 16 Upon
M.SssI binding to Y+CG⁄ CGM or Y+(N)4CG⁄
C(N)4GM duplexes with the Y+ adducts flanking the
CpG recognition site on the 5¢ side, or positioned four
nucleotide residues distant from the CpG site on the 5¢
side, respectively, the fluorescence emission yield
increases by a factor of only 3
Overall, these results indicate that the fluorescence
properties of B[a]P-DNA in the complexes with MTases
strongly depend on the (+)-cis-B[a]P-N2-dG and
(+)-trans-B[a]P-N2-dG adduct stereochemistry and on
the location of the adduct in either the MTase
recogni-tion site or the flanking sequences
Binding of M.SssI and M.HhaI to
oligodeoxynucleotide duplexes containing
the (+)-cis-B[a]P-N2-dG adduct
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 Kd values of the M.SssI or M.HhaI
complexes with the oligodeoxynucleotide duplexes
con-taining the (+)-cis-B[a]P-N2-dG adduct, we used a
competition equilibrium binding assay In these
com-petition experiments, unlabeled B[a]PDE-modified and
32P-labeled GCG⁄ CGMref 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 Kd values for the B[a]PDE-modified X+CG⁄ CGC and GCX+⁄ CGC duplexes are 1.2–1.3 times smaller than for the unmodified parent GCG⁄ CGC duplex, and the Kd value for the GCX+⁄ CGM duplex is about the same
as for the parent GCG⁄ CGM duplex (Table 2) How-ever, a 4.7-fold reduction in the binding affinity was observed in the case of binding of the M.HhaI with the X+CG⁄ CGM duplex containing the
(+)-cis-B[a]P-N2-dG adduct on the 5¢-side of the target dC residue The binding of M.SssI to X+CG⁄ CGC and GCX+⁄ CGC is favored by a factor of 2 relative to the unmodified GCG⁄ CGC duplex In the case of M.SssI•X+CG⁄ CGM•AdoHcy and M.SssI•GCX+⁄ CGM•AdoHcy complexes, the Kdvalues are about the same as the Kd of the M.SssI•GCG ⁄ CGM•AdoHcy complex Thus, for both enzymes, the Kd 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 Kdvalues of the ternary complexes of MTases with the correspond-ing unmodified unmethylated (GCG⁄ CGC) or hemi-methylated (GCG⁄ CGM) duplexes
Steady-state kinetics of methylation of oligodeoxynucleotide duplexes containing (+)-cis-B[a]P-N2-dG adduct by M.SssI and M.HhaI The rates of methylation of the X+CG⁄ CGC, GCX+⁄ CGC, X+CG⁄ CGM and GCX+⁄ CGM duplexes by SssI and HhaI MTases were determined under steady-state conditions (Fig 4), and the kcat values were cal-culated (Table 2) The kcat values of methylation of B[a]PDE-modified unmethylated X+CG⁄ CGC and GCX+⁄ CGC duplexes or hemimethylated X+CG⁄ CGM and GCX+⁄ CGM duplexes by M.HhaI were decreased by factors of 1.2–1.7 in comparison with the
kcat 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 kcat for the hemimethylated X+CG⁄ CGM duplex containing (+)-cis-B[a]P-N2-dG on the 5¢-side of the target dC residue The kcat value for the hemimethylated duplex GCX+⁄ CGM was about the same as that for the GCG⁄ CGM duplex The kcat values determined for the unmethylated X+CG⁄ CGC and GCX+⁄ CGC duplexes were only 1.3 times smaller than kcat for the GCG⁄ CGC duplex In summary, the presence of the (+)-cis-B[a]P-N2-dG adduct practically does not affect
Trang 6DNA methylation rates catalyzed by either M.SssI or
M.HhaI The extent of the methylation is almost
independent of the position of the damaged guanine
residue X+
Discussion
The goal of our study was to elucidate the effect of
stereochemistry and adduct conformation of
(+)-cis-B[a]P-N2-dG and (+)-trans-B[a]P-N2-dG adducts on
DNA methylation by prokaryotic MTases SssI and
HhaI The conformations of the stereoisomeric B[a]
P-N2-dG adducts have been investigated in detail
(summarized in [6]) Briefly, the bulky pyrene-like
aro-matic ring system in the (+)-trans-B[a]P-N2-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-N2-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-N2-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 the DNA double helix into the M.HhaI active-site pocket [45] The flipped out cytosine forms contacts with the catalytic loop of the enzyme from the DNA minor-groove side The contacts of the amino-acid
A
B
Fig 3 Equilibrium competitive binding of duplexes containing (+)-cis-B[a]P-N 2 -dG adduct and unmodified duplexes, to the MTases SssI and HhaI Autoradiographs of EMSA of competitive binding of the unlabe-led B[a]PDE-modified X+CG ⁄ CGM and
32 P-labeled GCG ⁄ CGM ref duplexes to M.HhaI (A) and the unlabeled X + CG ⁄ CGC and32P-labeled GCG ⁄ CGM ref
duplexes to M.SssI (B) The concentrations of compet-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) with32P-labeled duplex GCG ⁄ CGM ref in the presence of increasing concentrations of the competitor duplexes GCG ⁄ CGC (j), X + CG⁄ CGC (m), GCX + ⁄ CGC (d), GCG ⁄ CGM (h),
X + CG ⁄ CGM (n) or GCX + ⁄ CGM (s) The relative fraction of bound32P-labeled DNA (R) is the ratio of the fraction of bound
32 P-labeled DNA in the presence of the competitor DNA (cpm bound ⁄ cpm total ) to the fraction of bound 32 P-labeled DNA in the absence of the competitor DNA (cpm bound ⁄ cpm total ).
Trang 7residues within the catalytic domain of the M.HhaI
with the DNA minor groove play an important role in
the methylation reaction The contacts of the M.HhaI
small domain with the DNA major groove are
respon-sible for the specific MTase-DNA recognition Similar
structural features are likely to be present in the
tern-ary M.SssI•DNA•AdoHcy complex, as suggested by a
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-N2-dG and
(+)-cis-B[a]P-N2-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-N2-dG and (+)-trans-B[a]P-N2-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-N2-dG and (+)-cis-B[a]P-N2-dG adducts are not too different, a conclusion that is supported by the similar fluorescence yields of the two types of adduct in the absence of protein (Fig 2A)
The fluorescence properties of the pyrenyl residues
in the B[a]P-N2-dG adducts are known to be sensitive
to their microenvironments [49,50], particularly in complexes with proteins [51,52] To elucidate possible differences in the microenvironments of the (+)-cis and (+)-trans adducts within MTase•B[a]P-DNA com-plexes, we examined changes in fluorescence intensities
of the two stereoisomeric B[a]P residues in the duplexes depicted in Tables 2 and 3 when the two dif-ferent MTases were added to aqueous solutions of these duplexes The enhancement in the fluorescence yield is substantially greater when MTase binds to oligonucleotide duplexes containing the (+)-trans adduct than the (+)-cis adduct (Fig 2) We observed
a 3.5–4-fold fluorescence increase upon M.HhaI bind-ing to duplexes containbind-ing the (+)-cis-B[a]P-N2-dG adduct and a 20–30-fold fluorescence enhancement upon the binding of M.HhaI to duplexes containing the stereoisomeric (+)-trans-B[a]P-N2-dG adduct A 1.6–1.7-fold fluorescence increase occurred upon M.SssI binding to the duplexes containing (+)-cis-B[a]P-N2-dG adduct and a 2.8–16-fold upon binding
of M.SssI to the duplexes containing
(+)-trans-B[a]P-N2-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-N2-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•GCY+⁄ 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
Fig 4 Steady-state kinetic analysis of methylation of unmodified
and (+)-cis-B[a]P-N2-dG adduct-containing oligodeoxynucleotide
duplexes by M.SssI (A) and M.HhaI (B) The concentrations of
GCG⁄ CGC, X + CG ⁄ CGC, GCX + ⁄ CGC and GCG ⁄ CGM duplexes were
250 n M , and the concentrations of X+CG ⁄ CGM and GCX + ⁄ CGM
duplexes were 350 n M Designations of unmodified and
B[a]PDE-modified duplexes on the curves are as shown in Fig 3.
Trang 8protein (Fig 2) Such enhancements are consistent
with the effects observed in the case of the nucleoside
(+)-trans-B[a]P-N2-dG adducts when water is replaced
by more hydrophobic organic solvents [53] Our
hypo-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-N2-dG adduct
than in the case of the (+)-trans-B[a]P-N2-dG adduct
Thus, the different microenvironment of the pyrenyl
residue in the (+)-cis-B[a]P-N2-dG adduct and
(+)-trans-B[a]P-N2-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-N2-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 fluorescdepend-ence 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 with GCY+⁄ CGM, Y+CG⁄ CGM and Y+(N)4CG⁄ C(N)4GM duplexes (Fig 2C) It is well established that neighboring bases in their normal positions in DNA quench the fluorescence of (+)-trans-B[a]P-N2
-dG introduced into oligodeoxynucleotide duplexes [39,55,56] We suggest that the observed large increase
in fluorescence in the case of the complex of M.SssI with the GCY+⁄ CGM duplex containing the (+)-trans adduct in the CpG site may be caused by diminished quenching by the target dC residue that is flipped in the MTase–DNA complex When the B[a]P residue is separated by four nucleotides from the target dC residue in the Y+(N)4CG⁄ C(N)4GM 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-N2-dG and (+)-cis-B[a]P-N2-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 The enzyme is shown in the ribbon repre-sentation DNA and AdoHcy are shown in the stick representation.
Trang 9enhancement is also small We showed previously that
methylation of this duplex was essentially inhibited,
even under single-turnover conditions for 2 h, and it
was assumed that the flipping of the target base was
impeded [37] Therefore, in this case there was
prob-ably no effect of the flipping of the target cytosine on
the B[a]P fluorescence Overall, the changes in
fluores-cence intensities are clearly due to changes in the local
microenvironment of the B[a]P residues in the DNA
duplexes, and are consistent with a base-flipping model
of the dC target residue
Binding and methylation studies
The interactions of M.HhaI and M.SssI with DNA
containing site-specifically positioned
(+)-trans-B[a]P-N2-dG adduct have recently been investigated [37] The
Kd and kcat values for the (+)-cis-B[a]P-N2-dG and
(+)-trans-B[a]P-N2-dG adducts in different sequence
contexts are compared with one another in Fig 6 The
minor-groove position of the (+)-trans-B[a]P-N2-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-N2-dG into DNA does not cause any signifi-cant changes in Kd 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-N2-dG adducts in DNA does not influence DNA binding in the case of M.SssI, but, in contrast, does affect DNA binding in the case of M.HhaI The (+)-trans-B[a]P-N2-dG adduct greatly dimin-ishes the methylating efficiency of hemimethylated (by factors of 185–5000) and unmethylated (by factors of 1.3–9) DNA catalyzed by either M.SssI or M.HhaI [37] when the (+)-trans-B[a]P-N2-dG adduct is positioned 5¢ to the target dC base (Fig 6) On the other hand, the (+)-cis-B[a]P-N2-dG adduct has practically no effect on the methylation rate constant, kcat, in either case (Table 2, Fig 6) These differences are a direct consequence of the strikingly different conformational characteristics of the stereoisomeric (+)-cis-B[a]P-N2
-dG and (+)-trans-B[a]P-N2-dG adducts It is likely that, in the (+)-trans-B[a]P-N2-dG adduct, the bulky B[a]P residue situated in the minor groove interferes 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-N2-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 kcat(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 Kreld and krelcat
values for duplexes containing the
(+)-cis-B[a]P-N 2 -dG adduct were calculated relative
to the canonical, unmodified duplex
GCG⁄ CGM from the data presented in
Table 2 The Kreld and krelcatvalues for
duplexes containing the (+)-trans-B[a]P-N2
-dG adduct were calculated in a similar way
from the data presented in [37] G* is
(+)-cis-B[a]P-N2-dG (X+) or (+)-trans-B[a]P-N2-dG
(Y + ) The target dC residue is underlined.
Trang 10helix, 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 Kd and kcat 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 GCG⁄ 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, kcatremains unchanged when the (+)-cis adduct
in the GCX+⁄ CGM duplex is positioned on the 3¢-side
of the target dC residue In the case of M.HhaI, the
kcatvalues are practically unchanged in the presence of
the (+)-cis adduct in both hemimethylated duplexes
(Fig 6)
Conclusions
In contrast with the (+)-trans-B[a]P-N2-dG adduct
[37], the introduction of the stereoisomeric
(+)-cis-B[a]P-N2-dG adduct into the DNA recognition sites of
the prokaryotic MTases M.HhaI and M.SssI does not
have any significant effect on DNA methylation rates
This difference may be associated with the intercalative
conformation of the (+)-cis adduct and the
minor-groove conformation of the (+)-trans adduct, the
lat-ter inlat-terfering with inlat-teractions of the catalytic loops
of the MTases and the minor groove of DNA In
accordance with this hypothesis, the fluorescence
prop-erties of the pyrenyl residues of the (+)-cis-B[a]P-N2
-dG or (+)-trans-B[a]P-N2-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) [CH3-3H]AdoMet (77 CiÆmmol)1,
13 lm) was from Amersham Biosciences (Little Chalfont,
UK) [c-32P]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 His6-tagged M.SssI (6.7 lm) To obtain His6-tagged M.SssI, an appropriate hybrid plasmid was produced [58] using the vector pCAL7 provided by New England BioLabs (Beverly, MA, USA) The kinetic parameters determined with wild type or His6 -tagged M.SssI were practically identical in value The MTases were found to be homogeneous on 12% polyacryl-amide gels in the presence of 0.1% SDS T4 polynucleotide kinase was obtained from MBI Fermentas (Vilnius, Lithu-ania) Buffers A–F were prepared using Milli-Q water:
A, 10 mm Tris•HCl (pH 7.9) ⁄ 50 mm NaCl; B, buffer A containing 1 mm dithiothreitol; C, buffer B, containing 0.1 mgÆmL)1 acetylated BSA; D, 50 mm Tris•HCl (pH 7.5)⁄ 50 mm NaCl ⁄ 10 mm EDTA ⁄ 5 mm 2-mercaptoeth-anol; E, 50 mm Tris•HCl (pH 7.5) ⁄ 50 mm NaCl ⁄ 10 mm EDTA⁄ 5 mm 2-mercaptoethanol ⁄ 0.2 mgÆmL)1 acetylated BSA; F, 50 mm Tris•H3BO3(pH 8.3)⁄ 2 mm EDTA
Oligodeoxynucleotides
The sequences of the oligodeoxynucleotides used are sum-marized in Table 1 GCGref, CGMref, 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-N2-dG adduct were obtained as des-cribed [49] The site-specifically modified X+CG and GCX+ oligodeoxynucleotides containing a single (+)-cis-B[a]P-N2-dG lesion were obtained by treatment of GCG with racemic B[a]PDE solution using previously described methods [59] The (+)-trans-B[a]P-N2-dG, (–)-trans-B[a] P-N2-dG and (+)-cis-B[a]P-N2-dG adducts at the 18-mer oligodeoxynucleotide level were separated and purified by reverse-phase HPLC on an X Terra C18 column (Waters, Milford, MA, USA) [59]
All oligodeoxynucleotides were further purified by elec-trophoresis on denaturing 20% polyacrylamide gels and desalted by passing the solutions through C18 September-Pack cartridges (Waters) The sequences were labeled by the standard 32P-5¢-phosphorylation of oligodeoxynucleo-tides using T4 polynucleotide kinase and [c-32P]ATP Oligodeoxynucleotide concentrations were estimated spec-trophotometrically The absorption coefficients of unmodi-fied and B[a]PDE-modiunmodi-fied oligonucleotides were calculated
as described [36]
Fluorescence measurements
The fluorescence of the X+CG⁄ CGM, GCX+⁄ CGM,
Y+CG⁄ CGM, GCY+⁄ CGM and Y+(N)4CG⁄ C (N)4GM 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⁄