Tài liệu Báo cáo khoa học: The stereochemistry of benzo[a]pyrene-2¢-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases pptx

The stereochemistry of benzo[a]pyrene-2¢-deoxyguanosine adducts affects DNA methylation by SssI and HhaI DNA methyltransferases Oksana M Subach1, Diana V Maltseva1, Anant Shastry2, Alexander Kolbanovskiy2, ˇ Saulius Klimasauskas3, Nicholas E Geacintov2 and Elizaveta S Gromova1 Chemistry Department, Moscow State University, Russia Department of Chemistry, New York University, NY, USA Laboratory of Biological DNA Modification, Institute of Biotechnology, Vilnius, Lithuania 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 basedisplaced (+)-cis adduct does not interfere with the minor-groove DNA– catalytic loop contacts, allowing near-normal binding of the MTases and undiminished kcat values The polycyclic aromatic hydrocarbons are a wellknown 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 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-epoxy7,8,9,10-tetrahydrobenzo[a]pyrene; B[a]P-DNA, DNA containing benzo[a]pyrene; C5 MTase, C5-cytosine DNA methyltransferase; EMSA, electrophoretic mobility shift assay; kcat, multiple turnover rate constant; Kd, 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 FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2121 Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al O N NH N N R NH HO HO OH Y+ - (+)-trans-B[a]P-N2-dG O N N R NH N NH HO HO OH X+ - (+)-cis-B[a]P-N2-dG Fig Chemical structures of the (+)-trans-B[a]P-N2-dG and (+)-cisB[a]P-N2-dG adducts {(+)-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 (+)-cisB[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 (+)-cisB[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 characteristics of the (–)-trans-B[a]P-N2-dG and (+)-transB[a]P-N2-dG adducts [6] The structures of (+)-trans-B[a]P-N2-dG and (+)-cisB[a]P-N2-dG adducts in dsDNA are very different from one another, the former being characterized by 2122 an external minor-groove conformation and the latter by a base-displaced intercalative conformation [6,9,10] The different structural characteristics have a pronounced effect on the cellular processing of these stereoisomeric 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, transcription, and the interaction of different proteins with DNA All four B[a]P-N2-dG adducts inhibit DNA replication [13,14] The successful, although error-prone, translesional synthesis past both stereoisomeric adducts has been reported [13,15,16] The fidelity of translesional 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 (+)-transB[a]P-N2-dG adduct than by the (+)-cis-B[a]P-N2-dG adduct [17] The binding of the transcription factor Sp 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 transcription factor to DNA containing different stereoisomeric 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 (+)-cisB[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 conformations of (+)-trans-B[a]P-N2-dG and (+)-cis-B[a]PN2-dG lesions DNA methylation plays an important role in different cellular processes such as regulation of transcription, cell development, and chromatin structure [22,23] Mammalian genomes are methylated at certain 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 models 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 mammalian MTases [30] FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al B[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 normal 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 absolute 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 EcoRII [36], SssI, and HhaI [37] was examined In most cases, the methylation efficiency of oligodeoxynucleotide duplexes containing trans adducts in MTase recognition 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 contacts [37] Because of the markedly different (+)-trans-B[a]PN2-dG and (+)-cis-B[a]P-N2-dG adduct conformations, 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-antiB[a]P-N2-dG adduct on the DNA binding and catalytic activity of SssI and HhaI was examined and compared Table Oligodeoxynucleotide sequences synthesized M, m5dC; X+, (+)-cis-B[a]P-N2-dG; Y+, (+)-trans-B[a]P-N2-dG GCGref CGMref GCG CGC CGM X+CG GCX+ Y+CG GCY+ 5¢-GAGCCAAGCGCACTCTGA 5¢-TCAGAGTGMGCTTGGCTC 5¢-CACCCTTGCGCTCTCTCA 5¢-TGAGAGAGCGCAAGGGTG 5¢-TGAGAGAGMGCAAGGGTG 5¢-CACCCTTX+CGCTCTCTCA 5¢-CACCCTTGCX+CTCTCTCA 5¢-GAGCCAAY+CGCACTCTGA 5¢-GAGCCAAGCY+CACTCTGA 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 spectroscopic methods, we show here that the (+)-cis-antiB[a]P-N2-dG adducts indeed not significantly inhibit methylation, demonstrating that the stereochemistry of B[a]P metabolite-derived DNA adducts can affect this potentially important epigenetic mechanism of cancer initiation [1,38] Results The (+)-cis-B[a]P-N2-dG lesions (X+) were site-specifically incorporated into the single-stranded oligonucleotides shown in Table The corresponding duplexes are shown in Table The X+ residues were introduced into the overlapping recognition sites of both M.SssI (CpG) and M.HhaI (GCGC) on either the Table Properties of the oligodeoxynucleotide duplexes containing (+)-cis-B[a]P-N2-dG adduct as substrates of M.HhaI and M.SssI The target dC are underlined M.SssI ⁄ M.HhaI sites are in bold The other designations are as in Table M.HhaI M.SssI Designation DNA duplex Kd (pM) kcat (min)1) Kd (nM) GCG ⁄ CGC 5¢-CACCCTTGCGCTCTCTCA 3¢-GTGGGAACGCGAGAGAGT + 5¢-CACCCTTX CGCTCTCTCA 3¢-GTGGGAAC GCGAGAGAGT + 5¢-CACCCTTGCX CTCTCTCA 3¢-GTGGGAACGC GAGAGAGT 5¢-CACCCTTGCGCTCTCTCA 3¢-GTGGGAACGMGAGAGAGT + 5¢-CACCCTTX CGCTCTCTCA 3¢-GTGGGAAC GMGAGAGAGT + 5¢-CACCCTTGCX CTCTCTCA 3¢-GTGGGAACGM GAGAGAGT 52.7 ± 1.3 3.4 ± 0.4 6.8 ± 1.2 0.9 ± 0.4 41.6 ± 1.2 2.7 ± 0.3 3.0 ± 0.3 0.7 ± 0.3 42.8 ± 13.9 2.0 ± 0.2 3.8 ± 0.6 0.7 ± 0.2 13.0 ± 3.9 2.1 ± 0.3 4.1 ± 0.8 0.4 ± 0.2 61 ± 14 1.5 ± 0.3 4.2 ± 0.5 0.13 ± 0.05 13.6 ± 2.5 1.7 ± 0.2 1.9 ± 0.2 0.5 ± 0.1 X+CG ⁄ CGC GCX+ ⁄ CGC GCG ⁄ CGM X+CG ⁄ CGM GCX+ ⁄ CGM FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS kcat (min)1) 2123 Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al 5¢- or the 3¢-side of the target dC residue In hemimethylated 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 hemimethylated X+CG ⁄ CGM and GCX+ ⁄ CGM duplexes containing (+)-cis-B[a]P-N2-dG were cooperative with the melting temperature ranging from 57 to 61 °C, being 4–8 °C 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 conformational 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 oligodeoxynucleotide duplexes were titrated with various amounts of M.HhaI or M.SssI The duplexes containing Y+ are defined in Table The Y+ residues were introduced 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 duplexes alone or in complexes with MTases exhibit the usual broad maxima at 384 and 404 nm (Fig 2A), consistent 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 Oligodeoxynucleotide duplexes containing (+)-trans-B[a] P-N2-dG adduct N is any nucleotide residue The other designations are as in Tables and Designation DNA duplex GCG ⁄ CGM 5¢-GAGCCAAGCGCACTCTGA 3¢-CTCGGTTCGMGTGAGACT + 5¢-GAGCCAAY CGCACTCTGA 3¢-CTCGGTTC GMGTGAGACT + 5¢-GAGCCAAGCY CACTCTGA 3¢-CTCGGTTCGM GTGAGACT 5¢-GCTY+GTGGCGTAGGC 3¢-CGAC CACCGMATCCG Y+CG ⁄ CGM GCY+ ⁄ CGM Y+(N)4CG ⁄ C(N)4GM 2124 Fig Fluorescence titration of DNA containing (+)-cis-B[a]P-N2-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]PDNA•AdoHcy complexes and the free B[a]P-DNA duplexes; 500 nM GCX+ ⁄ CGM or GCY+ ⁄ CGM duplexes were incubated with 875 nM M.HhaI in the presence of 0.1 mM AdoHcy in buffer D The fluorescence excitation wavelength was 350 nm (B) 500 nM GCX+ ⁄ CGM (s), GCY+ ⁄ 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 nM GCX+ ⁄ CGM (s), X+CG ⁄ CGM (n), Y+CG ⁄ CGM (j), GCY+ ⁄ CGM (r) or Y+(N)4CG ⁄ C(N)4GM (·) were titrated with M.SssI in buffer B at 25 °C The excitation and emission wavelengths are the same as in (B) FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al emission 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 fluorescence 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-N2dG adduct in the recognition site or in the flanking sequence leads to a 1.6–1.7-fold increase in the fluorescence emission intensity of the B[a]P residue (Fig 2C) The fluorescence intensity of the B[a]P residue increases 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 (+)-transB[a]P-N2-dG adducts in the recognition site, the fluorescence 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  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 recognition 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 oligodeoxynucleotide duplexes was performed in the presence of the cofactor analog S-adenosyl-l-homocysteine (AdoHcy) 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 containing the (+)-cis-B[a]P-N2-dG adduct, we used a competition equilibrium binding assay In these competition experiments, unlabeled B[a]PDE-modified and 32 P-labeled GCG ⁄ CGMref duplexes were mixed before the addition of MTase The formation of the complexes 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) However, 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]PN2-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  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 Kd values 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 Kd values of the ternary complexes of MTases with the corresponding unmodified unmethylated (GCG ⁄ CGC) or hemimethylated (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 steadystate conditions (Fig 4), and the kcat values were calculated (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 FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2125 Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al A Fig Equilibrium competitive binding of duplexes containing (+)-cis-B[a]P-N2-dG adduct and unmodified duplexes, to the MTases SssI and HhaI Autoradiographs of EMSA of competitive binding of the unlabeled B[a]PDE-modified X+CG ⁄ CGM and 32 P-labeled GCG ⁄ CGMref duplexes to M.HhaI (A) and the unlabeled X+CG ⁄ CGC and 32P-labeled GCG ⁄ CGMref duplexes to M.SssI (B) The concentrations of competitor X+CG ⁄ CGM were 0, 0.5, 1, 5, 10, 40, 80, 120, 200 nM in lanes 1–9, respectively (A), and 0, 10, 20, 50, 100, 200, 300, 400, 500 nM in lanes 1–9, respectively (B) Equilibrium competition curves for complexes of M.HhaI (C) and M.SssI (D) with 32P-labeled duplex GCG ⁄ CGMref 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 bound 32P-labeled DNA (R) is the ratio of the fraction of bound 32 P-labeled DNA in the presence of the competitor DNA (cpmbound ⁄ cpmtotal) to the fraction of bound 32P-labeled DNA in the absence of the competitor DNA (cpmbound° ⁄ cpmtotal°) B C D DNA 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 (+)-cisB[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 aromatic 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 conformation with the modified dG residue and the partner 2126 base dC in the opposite strand displaced into the minor and major grooves, respectively [9] Molecular views of these (+)-cis and (+)-trans adduct conformations are shown in Fig 5A The structure of the MTase–DNA complexes containing 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 containing 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 FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al A B Fig 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 nM, and the concentrations of X+CG ⁄ CGM and GCX+ ⁄ CGM duplexes were 350 nM Designations of unmodified and B[a]PDEmodified duplexes on the curves are as shown in Fig residues 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 responsible for the specific MTase-DNA recognition Similar structural features are likely to be present in the ternary 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 solutions [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 within 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, unpublished) Thus, in the absence of protein, the fluorescence 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 complexes, we examined changes in fluorescence intensities of the two stereoisomeric B[a]P residues in the duplexes depicted in Tables and when the two different 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 binding to duplexes containing 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 (+)-cisB[a]P-N2-dG adduct and a 2.8–16-fold upon binding of M.SssI to the duplexes containing (+)-trans-B[a]PN2-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 residues in the protein–DNA complexes It is known from previous studies that the fluorescence 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 suggest that the (+)-trans adducts are situated in a more hydrophobic environment in the protein complexes than in aqueous solution in the absence of FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2127 Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al A B Fig (A) Conformations of the B[a]PDEmodified duplexes containing the (+)-transB[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 cytosine, and AdoHcy are depicted in dark grey The enzyme is shown in the ribbon representation DNA and AdoHcy are shown in the stick representation protein (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 hypothesis is that the change in the hydrophobicity of the local environment upon protein binding is less pronounced 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 complexes is revealed by fluorescence studies It has been postulated that the flipping or extrusion of the target base from the DNA duplex is an important intermediate step in DNA methylation catalyzed by C5 MTases [54] We postulated that the fluorescence 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 depend2128 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 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-N2dG 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 duplex, 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 FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al enhancement is also small We showed previously that methylation of this duplex was essentially inhibited, even under single-turnover conditions for h, and it was assumed that the flipping of the target base was impeded [37] Therefore, in this case there was probably no effect of the flipping of the target cytosine on the B[a]P fluorescence Overall, the changes in fluorescence 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]PN2-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 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 minorgroove DNA–M.HhaI contacts and does not significantly 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 significant 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 residues in the (+)-cis adducts which interferes less significantly 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 diminishes 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-N2dG 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 oligodeoxynucleotide 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 Fig Bar graphs representing relative Kd (K rel ) and kcat (k rel ) values for binding and d cat methylation of DNA containing (+)-cis-B[a]PN2-dG and (+)-trans-B[a]P-N2-dG adducts by M.SssI (A) and M.HhaI (B) The K rel and k rel d cat values for duplexes containing the (+)-cisB[a]P-N2-dG adduct were calculated relative to the canonical, unmodified duplex GCG ⁄ CGM from the data presented in Table The K rel and k rel values for d cat duplexes containing the (+)-trans-B[a]P-N2dG 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 B FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2129 Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al 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 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, kcat remains 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 kcat values are practically unchanged in the presence of the (+)-cis adduct in both hemimethylated duplexes (Fig 6) 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 His6tagged M.SssI were practically identical in value The MTases were found to be homogeneous on 12% polyacrylamide gels in the presence of 0.1% SDS T4 polynucleotide kinase was obtained from MBI Fermentas (Vilnius, Lithuania) Buffers A–F were prepared using Milli-Q water: A, 10 mm Tris•HCl (pH 7.9) ⁄ 50 mm NaCl; B, buffer A containing mm dithiothreitol; C, buffer B, containing 0.1 mgỈmL)1 acetylated BSA; D, 50 mm Tris•HCl (pH 7.5) ⁄ 50 mm NaCl ⁄ 10 mm EDTA ⁄ mm 2-mercaptoethanol; E, 50 mm Tris•HCl (pH 7.5) ⁄ 50 mm NaCl ⁄ 10 mm EDTA ⁄ mm 2-mercaptoethanol ⁄ 0.2 mgặmL)1 acetylated BSA; F, 50 mm TrisãH3BO3 (pH 8.3) mm EDTA Oligodeoxynucleotides In contrast with the (+)-trans-B[a]P-N2-dG adduct [37], the introduction of the stereoisomeric (+)-cisB[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 minorgroove conformation of the (+)-trans adduct, the latter interfering with interactions of the catalytic loops of the MTases and the minor groove of DNA In accordance with this hypothesis, the fluorescence properties of the pyrenyl residues of the (+)-cis-B[a]P-N2dG 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 mammalian MTases, and these possibilities are being investigated in our laboratory The sequences of the oligodeoxynucleotides used are summarized in Table 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 described [49] The site-specifically modified X+CG and GCX+ oligodeoxynucleotides containing a single (+)-cisB[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 electrophoresis on denaturing 20% polyacrylamide gels and desalted by passing the solutions through C18 SeptemberPack cartridges (Waters) The sequences were labeled by the standard 32P-5¢-phosphorylation of oligodeoxynucleotides using T4 polynucleotide kinase and [c-32P]ATP Oligodeoxynucleotide concentrations were estimated spectrophotometrically The absorption coefficients of unmodified and B[a]PDE-modified oligonucleotides were calculated as described [36] Experimental procedures Fluorescence measurements Conclusions Chemicals and enzymes AdoMet and AdoHcy were purchased from Sigma (St Louis, MO, USA) [CH3-3H]AdoMet (77 CiỈmmol)1, 13 lm) was from Amersham Biosciences (Little Chalfont, UK) [c-32P]ATP (1000 CiỈmmol)1) was bought from Izotop (Obninsk, Russia) M.HhaI (4.4 mgỈmL)1) was prepared as 2130 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 spectrofluorimeter with slit widths of 5–10 nm for excitation and 3–5 nm for the emission monochromator All titrations were performed in a micro quartz cuvette (10 mm · 10 mm, 100 lL; Starna Cells, Atascadero, CA, USA) X+CG ⁄ FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al CGM, GCX+ ⁄ CGM, Y+CG ⁄ CGM, GCY+ ⁄ CGM or Y+(N)4CG ⁄ C (N)4GM duplexes were incubated with various concentrations of M.HhaI or M.SssI in buffer D or B in the presence of 100 lm AdoHcy at 25 °C for 10 The fluorescence emission spectra of the B[a]P-DNA adducts were measured at 360–480 nm with an excitation wavelength of 350 nm In separate control experiments, M.HhaI or M.SssI was incubated in buffer D or B, respectively, in the presence of 100 lm AdoHcy at 25 °C for 10 in the absence of B[a]P-DNA, and emission spectra were measured at 360–480 nm with excitation at 350 nm These control spectra were subtracted from those of the M.HhaI•B[a]P-DNA•AdoHcy or M.SssI•B[a]P-DNA•AdoHcy complexes For each oligodeoxynucleotide duplex alone and in complexes with MTases, the fluorescence intensities at 384 nm were calculated Determination of the amounts of the active form of M.SssI and M.HhaI The amount of the active form of M.SssI (338 ± 22 nm) and M.HhaI (120 ± 10 lm) was determined as described previously [37] Equilibrium competition experiments Determination of Kd by the EMSA method To obtain modified oligodeoxynucleotide duplexes X+CG ⁄ CGC, GCX+ ⁄ CGC, X+CG ⁄ CGM and GCX+ ⁄ CGM, the unmodified strands CGC or CGM were mixed with a twofold 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 not bind X+CG or GCX+ strands (data not shown) To obtain unmodified GCG ⁄ CGC and GCG ⁄ CGM duplexes, the oligodeoxynucleotide strands were mixed in the ratio : In the case of M.SssI, the reference 32P-labeled GCG ⁄ CGMref duplex (100 nm) was mixed with increasing concentrations of the B[a]PDEmodified competitor duplex (0–250 nm) in buffer C containing 8% glycerol and AdoHcy (1 mm) In the case of M.HhaI, the 32P-labeled GCG ⁄ CGMref duplex (2 nm) was mixed with increasing concentrations of the B[a]PDE-modified competitor duplex (0–90 nm) in buffer E containing 8% glycerol and AdoHcy (0.1 mm) M.SssI or M.HhaI was added to a final concentration of 50 nm or 0.6 nm, respectively, and samples were incubated at room temperature for and then at °C for 10 min, and were analyzed by nondenaturing 8% PAGE in 0.5 · buffer F When exposed, radioactive gels were processed as described in [37], and the values of k ¼ K r ⁄ Kd were determined as described in d [37], where K r and Kd are the dissociation constants of the d MTase•GCG ⁄ CGMref•AdoHcy and MTase•B[a]P-DNA• AdoHcy complexes The k ratio specifies the relative binding efficiency of the reference unmodified DNA and the competitor damaged DNA to the MTase The Kd values were calculated by dividing K r (see below) by the experid mental values of k Determination of Kr for the M.HhaI(M.SssI)• d GCG ⁄ CGMref•AdoHcy complexes We were unable to obtain an accurate K r values for the d M.HhaI•GCG ⁄ CGMref•AdoHcy complex by direct titration of solutions of the GCG ⁄ CGMref duplex with increasing enzyme concentrations This was due to the considerable experimental error associated with the dilution of the enzyme and the low radioactivity of the DNA at low (picomolar) concentrations The K r value for the d M.HhaI•GCG ⁄ CGMref•AdoHcy complex was calculated by multiplying the ratio k (obtained from the competitive binding of GCG ⁄ CGMref and Y+CG ⁄ CGM to M.HhaI) by the Kd value of the M.HhaI•Y+CG ⁄ CGM•AdoHcy complex The Kd value of the M.HhaI•Y+CG ⁄ CGM•AdoHcy complex was obtained by direct titration using EMSA The 32P-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 and at °C for 10 The further experimental procedures and data analysis were the same as described in [37] The K r value for d the M.SssI•GCG ⁄ CGMref•AdoHcy complex was determined 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 (CH3-3H) incorporated into the oligodeoxynucleotide duplexes [60] The reactions were carried out in buffer B for M.SssI, or buffer D for M.HhaI The mixtures contained GCG ⁄ CGC, X+CG ⁄ CGC, GCX+ ⁄ CGC, GCG ⁄ CGM, X+CG ⁄ CGM or GCX+ ⁄ CGM, M.SssI (17 nm) or M.HhaI (5 nm), and [CH3-3H]AdoMet (1.3 lm) In the case of both enzymes, the saturating duplex concentrations were found at which the V0 values were not changed These DNA concentrations were 0.25–0.35 lm or 0.05– 0.5 lm in the M.SssI and M.HhaI reactions, respectively The reactions were started by the addition of the MTase After 0.5–15 of incubation at 37 °C, aliquots of the reaction mixtures were pipetted on to DE-81 paper disks (Whatman, Brentford, UK) and treated as described [61] The amounts of methylated DNA were computed as described [60] The V0 values for all duplexes were determined from the initial linear portions of the product versus time profiles In FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS 2131 Stereochemistry of B[a]P-N2-dG affects methylation O M Subach et al the used duplex concentration ranges, the measured V0 values were constant for each unmodified or B[a]PDE-modified duplex, indicating that the Vmax limit was reached Using the Vmax values thus obtained, the kcat values were calculated (Table 2) Acknowledgements This research was supported by a US Public Health Service grant No TW05689 from the Fogarty International Center (New York University and Moscow State University), NIH Grant CA 099194 (New York University), and RFBR Grants 04-04-49488 and 05-0449690 (Moscow State University) We thank Dr B Jack and Dr G Wilson from New England Biolabs for their gift of the M.SssI plasmid, Dr F Johnson and Dr R Bonala for synthesis of trans-B[a]P-N2-dG adducts, Dr N N Veiko and Dr N A Cherepanova for assistance with the fluorescence experiments 10 11 References Luch A (2005) Nature and nurture: lessons from chemical carcinogenesis Nat Rev Cancer 5, 113–125 Wei SJ, Chang RL, Hennig E, Cui XX, Merkler KA, Wong CQ, Yagi H, Jerina DM & 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methyltransferase–substrate interaction Eur J Biochem 271, 2391–2399 Huang W, Amin S & Geacintov NE (2002) Fluorescence characteristics of site-specific and stereochemically distinct benzo[a]pyrene diol epoxide-DNA adducts as probes of adduct conformation Chem Res Toxicol 15, 118–126 O’Gara M, Klimasauskas S, Roberts RJ & Cheng X (1996) Enzymatic C5-cytosine methylation of DNA: mechanistic implications of new crystal structures for HhaI methyltransferase-DNA-AdoHcy complexes J Mol Biol 261, 634–645 FEBS Journal 274 (2007) 2121–2134 ª 2007 The Authors Journal compilation ª 2007 FEBS ... 32P-labeled DNA in the absence of the competitor DNA (cpmbound° ⁄ cpmtotal°) B C D DNA methylation rates catalyzed by either M .SssI or M .HhaI The extent of the methylation is almost independent of the. .. compared with the levels of DNA methylation, and thus the effect of the absolute configurations and conformations of the adducts on DNA methylation was not evaluated More recently, the effect of stereochemically... 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 steadystate conditions (Fig 4), and