2-Pyrimidinone as a probe for studying the Eco RII DNA methyltransferase–substrate interaction Oksana M. Subach 1 , Anton V. Khoroshaev 1 , Dmitrii N. Gerasimov 1 , Vladimir B. Baskunov 1 , Anna K. Shchyolkina 2 and Elizaveta S. Gromova 1 1 Chemistry Department, Moscow State University, Russia; 2 Engelghardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5¢…CC*T/AGG…3¢ DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl- L -methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH 2 group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5¢…CCT/AGG…3¢ sequence repla- cing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal dena- turation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methy- lation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA–M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.Eco- RII. In the absence of the cofactor S-adenosyl- L -methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl- L -methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs. Keywords: 2-pyrimidinone; M.EcoRII; C5 cytosine DNA methyltransferase; inhibition; DNA recognition. DNA (cytosine-5)-methyltransferases (C5 MTases) catalyze the transfer of a methyl group from S-adenosyl- L -methio- nine (AdoMet) to cytosine C5 atom in specific DNA sequences. The methylation reaction of C5 MTases occurs with the addition of a cysteine thiol group from the conserved Pro-Cys motif to the C6 position of the target cytosine, followed by methyl transfer from AdoMet to the C5 position of the target base and the release of the methylated substrate [1,2] (Fig. 1A). It is important to note that the target cytosine is flipped out of the DNA double helix into the catalytic pocket of the enzyme and brought into proximity of the cofactor [2]. Several cytosine analogues, 5-fluorocytosine (FC), 5-azacytosine (AzaC) and 2-pyrimidinone (2P), have been reported as mechanism-based inhibitors of C5 MTases [3–6]. Introduction of a fluorine atom to the C5 position of the target cytosine results in an irreversible covalent attack of a cysteine residue and transfer of a methyl group to the C5 position of the target base [1]. Replacement of C5 by a nitrogen atom in azacytosine (AzaC) facilitates nucleophilic attack of the cysteine residue at the C6 position which occurs in the presence or absence of AdoMet [5]. Methyl or proton transfer to the N5 position occurs in the presence or absence of AdoMet, accordingly. As a result, two structures of the end product are possible: the enzyme linked to methylated AzaC or the enzyme linked to protonated AzaC [5].AsthereisnoprotonatC5totakeawaywhentheN5 position becomes methylated an irreversible covalent com- plex is formed with the enzyme. 2-Pyrimidinone (2P) is a cytosine analogue in which the exocyclic amino group is replaced by a hydrogen atom. Removal of the exocyclic amino group from the cytosine results in an increase of reactivity at the C6 carbon atom in 2P and in a reduction of the energy barrier for base flipping [7]. 2P replacing the target cytosine in the recognition sequences for HhaI[6],MspIandHgaI-2 [7–9] C5 MTases evokes covalent bond formation with these MTases. Zhou et al. [6] suggested the pathway of inhibition of C5 MTases by 2P-containing DNA in the absence of AdoMet (Fig. 1A). The reaction mechanism involves the addition of a cysteine thiol group of the enzyme (from conserved Pro-Cys motif) to the C6 position of 2-pyrimidinone followed by proton transfer to the C5 position. Due to the absence of the exocyclic amino group, b-elimination of the proton from the C5 position is retarded. The mechanism of inhibition of C5 MTases by 2P-containing DNA in the Correspondence to E. S. Gromova; Chemistry Department, Moscow State University, Moscow, 119992, Russia. Fax: + 7 095 939 31 81, Tel.: + 7 095 939 31 44, E-mail: gromova@genebee.msu.ru Abbreviations: C5 MTase, C5 cytosine DNA methyltransferase; FC, 5-fluorocytosine; AzaC, 5-azacytosine; M.EcoRII, EcoRII DNA methyltransferase; AdoMet, S-adenosyl- L -methionine; AdoHcy, S-adenosyl- L -homocysteine; 2P, 2-pyrimidinone. Enzyme: EcoRII DNA methyltransferase (EC 2.1.1.37). (Received 18 November 2003, revised 19 February 2004, accepted 14 April 2004) Eur. J. Biochem. 271, 2391–2399 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04158.x presence of AdoMet was not investigated. It should be noted that 1-(b- D -ribofuranosyl)-2-pyrimidinone, often referred to as zebularine, was used in vivo as an antitumor drug [10]. By now it is clear that its antitumor properties are likely attributed to inhibition of C5 MTase activity in tumor cells. 2-Pyrimidinone could also be considered as a mimic of an intermediate in the minor deaminative pathway of C5 MTases catalysis [6]. EcoRII DNA methyltransferase (M.EcoRII) catalyzes the transfer of a methyl group from AdoMet to the C5 position of the inner deoxycytidine residue (C*) in the DNA sequence 5¢…CC*T/AGG…3¢. It has been shown that M.EcoRII forms an irreversible covalent complex with DNA containing FC instead of the target cytosine [11,12]. Introduction of AzaC in this position also led to inhibition of the enzyme [5]. The mechanism of inhibition of M.Eco- RII by 2P-containing DNA is still unknown. Also, a study of the contributions of different functional groups of the 5¢…CCT/AGG…3¢ sequence to specific interaction with M.EcoRII is at the very beginning [13]. We aim to explore the effect of 2P substitution for C and T within the recognition sequence of M.EcoRII on recog- nition and catalysis performed by M.EcoRII. In this work, we have examined a DNA duplex containing 2-pyrimidi- none instead of the target cytosine as a potential inhibitor of M.EcoRII, and also the role of AdoMet in the formation of the covalent adduct between 2P-substituted DNA and M.EcoRII. Furthermore, we have studied the contribution of the functional groups of pyrimidine residues in DNA– M.EcoRII recognition by using duplexes containing 2-pyrimidinone in place of each pyrimidine base in the 5¢…CCT/AGG…3¢ sequence. Materials and methods Chemicals and enzymes AdoMet and AdoHcy were from Sigma (USA). [CH 3 – 3 H]AdoMet (77 CiÆmmol )1 ) was from Amersham (USA). [ 32 P]ATP[cP] (1000 CiÆmmol )1 ) was from Izotop (Obninsk, Russia). DNA-methyltransferase EcoRII (stock solution, 52 l M ) was overexpressed as an N-terminally His 6 -tagged protein and chromatographically purified on a nickel chelate column as described previously [14]. T4 polynucleotide kinase was from MBI Fermentas (Vilnius, Lithuania). Oligonucleotides Oligodeoxyribonucleotides containing 2-pyrimidinone were synthesized as described previously [15]. 32 P-phosphory- lation of the oligonucleotides was performed using T4-polynucleotide kinase and [ 32 P]ATP[cP]. UV thermal denaturation and thermodynamic parameters of duplex formation Heating of the samples containing 2.25 l M of duplexes in buffer A (40 m M Tris/HCl, pH 7.9, 1 m M EDTA, 50 m M NaCl) at temperatures ranging from 15 to 85 °Cwas performed at a constant rate of 0.2 °CÆmin )1 . Absorbance of duplexes at 260 nm was measured using Cary 50 Bio spectrophotometer (Varian, Victoria, Australia) with tem- perature controller. Thermodynamic analysis of helix-coil transition curves was performed using a two-state model. The thermodynamic parameters were determined through a fitting procedure based on minimization of the integral mean square deviation between the theoretical transition curves and experimental absorbance data. Circular dichroism CD measurements of duplexes (2.25 l M ) in buffer A were performed with Mark V Jobin Yvon dichrograph (Paris, France) in 1 cm thermostated cells. Gel mobility-shift assay To determine the active enzyme concentration, 20 n M M.EcoRII was incubated with 1–50 n M 32 P-labeled DNA duplex I¢ in 20 lL of buffer B (40 m M Tris/HCl, pH 7.9, 5m M dithiothreitol, 1 m M EDTA) containing 50 m M NaCl, 6% glycerol and 1 m M AdoHcy for 15 min at room temperature and 10 min at 0 °C. The reaction mixtures Fig. 1. Proposed mechanism of inhibition of M.EcoRII by 2P-containing DNA duplexes in the absence [6] (A) or in the presence (B) of AdoMet. In the case of M.EcoRII, the amino acid residue attacking the C6 position is Cys186 [11,12]; the general acid donating a proton to N3 is probably Glu233 [4,28]. 2392 O. M. Subach et al. (Eur. J. Biochem. 271) Ó FEBS 2004 were analysed by 8% native PAGE for 3 h at 120 V. The gel was prerun for 1 h at 100 V. Autoradiographs of the gels were prepared using Molecular Dynamics Phosphorimager (Amersham Biosciences, USA). Radioactivities of M.Eco- RII–DNA complex (cpm bound ) and free DNA (cpm free ) were determined. The ratio of bound to free DNA was calculated as (cpm bound )/(cpm free ); the concentration of bound DNA was calculated as [S 0 ][(cpm bound )/(cpm bound + cpm free )]. Data were analyzed by linear regression using the Microcal ORIGIN 6.0software.Forallfurtherexperiments the concentration of active form of M.EcoRII was used. To determine the apparent dissociation constants (K app d ), 1.5–150 n M M.EcoRII were incubated with 15 n M 32 P- labeled DNA duplexes I–IV in 15 lL of buffer B (40 m M Tris/HCl, pH 7.9, 5 m M dithiothreitol, 1 m M EDTA, con- taining 8% glycerol and 1 m M AdoHcy for 15 min at room temperature and 15 min at 0 °C. The reaction mixtures were processed as described above. Radioactivities of M.EcoRII– DNA complex (cpm bound ) and free DNA (cpm free )were determined and the fraction of bound DNA was calculated as (cpm bound )/(cpm bound +cpm free ). K app d was calculated by fitting the data to the following equation derived from a standard bimolecular binding equilibrium as described [16]: cpm bound ðcpm bound þcpm free Þ ¼ ½ES ½S 0  ¼ A 2½S 0  ð½S 0 þ½E 0 þK app d ÞÀ ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ð½S 0 þ½E 0 þK app d Þ 2 À4Á½E 0 Á½S 0  q  where [S 0 ] is total DNA concentration; [E 0 ]istotal M.EcoRII concentration; A is the factor accounting for nonideal equilibrium conditions during electrophoresis (cage effect, thermal dissociation). Nonlinear regression was performed using Microcal ORIGIN 6.0 software. Detection of DNA-enzyme adducts All reactions of 32 P-labeled duplexes I–III (100 n M )with M.EcoRII (200 n M ) were performed in 15 lL of buffer B containing 0.5 m M AdoMet or 0.5 m M AdoHcy. Reaction mixtures were incubated for 30 min at room temperature andfor15minoniceandprocessedbyoneofthefollowing ways: (a) incubated with 1% SDS at room temperature for 10 min and analyzed by 8% native PAGE; (b) incubated with 1% SDS at 65 °C for 5 min and analyzed by 8% native PAGE; (c) incubated with 0.8% SDS at room temperature for 10 min and analyzed by 12% SDS/PAGE (Laemmli gel). For autoradiography of the electrophoretic pattern, Kodak-XOMAT-S film was exposed with an intensifier screen at )20 °Covernight. Methylation assay For determination of initial methylation rates (V 0 ), methy- lation reactions were performed in 26 lL of buffer B, containing DNA duplexes I–IV or Im–IVm (1 l M ), M.Eco- RII (30 n M ), and [CH 3 – 3 H]-AdoMet (1.3 l M ). Reactions were started by adding the enzyme. After a 1, 2, 3, 4 and 5 min incubation at 15 °C, 5 lL of reaction mixture were pipetted onto DE81 (Whatman) paper disks. The disks were washed 5· for5minwith50m M KH 2 PO 4 ,3minwith water and 3 min with ethanol, air dried and placed into 5 mL of ZHS-106 scintillation fluid. The filter-bound radioactivity was measured on Tracor Analytic Delta 300 scintillation counter (ThermoQuest/CE Instruments, Piscataway, USA) and the amount of methylated DNA was determined as described [17]. Data were analyzed by linear regression using the Microcal ORIGIN 6.0 software. For quantification of the transfer of methyl groups to a 2-pyrimidinone residue in DNA duplexes by M.EcoRII methylation reactions were performed in 10 lL of buffer B, containing duplexes I, Im, III, IIIm or V (500–1000 n M ), M.EcoRII (12.5–1000 n M )and[CH 3 – 3 H]-AdoMet (1.3 l M ). In the case of M.HhaI methylation reactions were performed in 10 lL of buffer C (50 m M Tris/HCl, pH 7.5, 5m M 2-mercaptoethanol, 10 m M EDTA, 0.2 mg mL )1 bovine serum albumin), containing duplexes I, Vm or VIm (670 n M ), M.HhaI (20–16 000 n M )and[CH 3 – 3 H]-AdoMet (0.42 l M ). Reactions were started by adding the enzyme. After a 30 min incubation at 15 °C (reactions with M.Eco- RII) or at 20 °C (reactions with M.HhaI), 8 lL of reaction mixture were pipetted onto DE81 (Whatman) paper disks and processed as described for determination of V 0 . The time-dependent methyltransferase assays were per- formed by incubating M.EcoRII (500 n M ) with duplexes Im or IIIm (500 n M )and[CH 3 – 3 H]-AdoMet (1.3 l M ) in buffer Bat15°C for the indicated time periods. Reaction mixtures (8 lL) were pipetted onto DE81 (Whatman) paper disks and processed as described for determination of V 0 . Results and discussion To elucidate the mechanism of inhibition of M.EcoRII by 2-pyrimidinone modified DNA, and to understand the role of functional groups of pyrimidine bases of the recognition sequence in specific DNA–M.EcoRII inter- action, a series of 2P-containing substrate analogues was synthesized (duplexes II–IV, Table 1 and duplexes Iim–IVm, Table 2). Thermodynamics of formation of the 2P -containing DNA Insertion of 2P in place of C or T resulted in a marked destabilization of DNA duplexes [18–21]. To ascertain whether the 2P-containing DNA duplexes II–IV had a double helix structure under conditions of methylation by M.EcoRII, the thermodynamic stability of these duplexes was evaluated (Table 1). The values of the free energy of transition, as well as of the transition temperature (melting temperature, T m ), point to the following duplex stabilities: I > II > III > IV. In order to elucidate the contribution of a single nucleotide substitution, the value of the transition free energy of the nonmodified duplex I was subtracted from that of the modified duplexes II–IV (Table 1, DDG). The greatest energy penalty, DDG, for substitution of 2P for T in the TA base pair was 4.4 kcalÆmol )1 .The substitution of 2P for C appeared to be much more energetically disadvantageous in the base context C2PT (III) than in the context A2PC (II). The presence of only two H-bonds in the 2PG base pair vs. the three H-bonds in the CG base pair [19] as well as electrostatic interactions of 2P in the duplexes [21] may largely contribute to the destabilization of the duplexes. Ó FEBS 2004 2-Pyrimidinone as a probe for DNA methyltransferase (Eur. J. Biochem. 271) 2393 Conformation of the 2P -containing DNA To answer the question of whether 2P substitution for pyrimidines led to a marked distortion of DNA conforma- tion, we compared CD spectra of duplexes I–IV (Fig. 2). There are minor changes in the CD spectra, predominantly an intensity drop in the longwave CD band. This may be attributed to a strongly reduced absorption of the 2P base at 260 nm [18,19], which eliminates a potential contribution of the nearest-neighbor stacking contacts involving the 2PG or 2PA base pair to the CD signals in the corresponding spectral region. The difference in the CD spectra (Fig. 2, open symbols) reflects the decreased contribution of 2P to the conservative CD spectrum in comparison with the contributions of cytosine and thymine. Thus, the observed minor dissimilarity in the CD spectra of duplexes II–IV from CD spectrum of duplex I can be attributed to the distinctive optical features of the 2P analog, rather than to a marked distortion of the DNA conformation. Determination of concentration of active form of M.EcoRII It is known that the concentration of the active form of DNA methyltransferases does not correspond accurately to the total protein concentration [5,22]. The concentration of active form of M.EcoRII was estimated by titration of the enzyme (20 n M ) with M.EcoRII substrate in the presence of AdoHcy (Fig. 3, inset). The ratio of bound to free DNA was plotted vs. concentration of bound DNA (a Scatchard plot, Fig. 3) [23]. An 18-mer DNA duplex 5¢-GAG CCAACCTGGCTCTGA-3¢/3¢-CTCGGTTGGACCGAG ACT-5¢ (I¢) was used as M.EcoRII substrate. The horizontal axis intercept gives the total concentration of DNA binding sites (n[E 0 ]) equal to 2.35 ± 0.12 n M (Fig. 3) or 2.04 ± 0.25 (data not shown). As the number of DNA binding sites per molecule of enzyme (n) is 1 for M.EcoRII, the average concentration of M.EcoRII active form is 2.2 ± 0.2 n M . So, the amount of enzyme bound to DNA was assumed to be only 11 ± 1% of the total enzyme molecules. For all further experiments the concentration of active form of M.EcoRII was used. Binding and methylation of 2P -containing DNA by M.EcoRII Different heterocyclic base analogues have proven to be useful for studies of DNA–enzyme interactions [24]. In order to investigate the influence of introducing 2P into the different positions of the substrate DNA on the sequence- specific interaction of M.EcoRII with DNA, we studied Table 2. Relative initial methylation rates of hemimethylated 2P-con- taining DNA duplexes by M.EcoRII. Relative initial methylation rates (V rel 0 ) were calculated as ratio of V 0 of duplexes Iim–IVm to V 0 of duplex Im. M, 5-methylcytosine. Recognition sequence is in bold; 2P is underlined. No. DNA duplex V 0 (n M Æmin )1 )V rel 0 (%) Im 5¢-GCCAACCTGGCTCT-3¢/ 172.0 ± 34.4 100 ± 20 3¢-CGGTTGGAMCGAGA-5¢ IIm 5¢-GCCAA 2P CTGGCTCT-3¢/ 197.8 ± 53.3 115 ± 31 3¢-CGGTT-GGAMCGAGA-5¢ IIIm 5¢-GCCAAC 2P TGGCTCT-3¢/0 0 3¢-CGGTTG-GAMCGAGA-5¢ IVm 5¢-GCCAACC 2P GGCTCT-3¢/ 34.4 ± 3.4 20 ± 2 3¢-CGGTTGG-AMCGAGA-5¢ Fig. 2. CD spectra of DNA duplexes I–IV. ––,I;–d–, II; –m–, III; –j–, IV. Difference of CD spectra of I and duplexes: –s–, II; –n–, III; –h–, IV. Temperature was 20 °C. Table 1. Thermodynamic parameters of formation of 2P-containing DNA duplexes determined from thermal denaturation curves. Thermodynamic parameters and their standard deviations were determined from fitting the theoretical melting curves to experimental curves (see Materials and methods). Standard deviation of DS was less than 0.1 calÆmol )1 ÆK )1 . Experimental conditions see Materials and methods. 2P, 2-pyrimidinone; T m , melting temperature. Recognition sequence is in bold; 2P is underlined. No. DNA duplex T m (°C) DH (kcalÆmol )1 ) DS (kcalÆmol )1 ÆK )1 ) DG(T ¼ 20 °C) (kcalÆmol )1 ) DDG(T ¼ 20 °C) (kcalÆmol )1 ) I 5¢-GCCAACCTGGCTCT-3¢/ 52.6 ± 0.1 )65.0 ± 0.6 )173 )14.3 ± 0.6 – 3¢-CGGTTGGACCGAGA-5¢ II 5¢-GCCAA 2P CTGGCTCT-3¢/ 46.0 ± 0.1 )43.9 ± 0.7 )111 )11.5 ± 0.7 2.8 3¢-CGGTT-GGACCGAGA-5¢ III 5¢-GCCAAC 2P TGGCTCT-3¢/ 41.4 ± 0.1 )36.3 ± 0.4 )88 )10.3 ± 0.4 4 3¢-CGGTTG-GACCGAGA-5¢ IV 5¢-GCCAACC 2P GGCTCT-3¢/ 35.4 ± 0.1 )41.7 ± 0.7 )106 )9.9 ± 0.7 4.4 3¢-CGGTTGG-ACCGAGA-5¢ 2394 O. M. Subach et al. (Eur. J. Biochem. 271) Ó FEBS 2004 binding and methylation of canonical (I) and the 2P- containing DNA duplexes (II-IV) with M.EcoRII (Table 3). The formation of complexes was monitored by gel mobility- shift assays in the presence of AdoHcy because of the known increase in the affinity of M.EcoRII for DNA in the presence of the cofactor [12]. DNA duplexes were incubated with increasing M.EcoRII concentrations at saturating AdoHcy concentrations. A binding isotherm and corres- ponding autoradiograph of a typical experiment are shown in Fig. 4. The calculated apparent dissociation constants (K app d ) are summarized in Table 3. The methylation reactions were performed under steady- state conditions (Tables 2 and 3). Reactions of M.EcoRII with hemimethylated duplexes Im–IVm were performed in order to determine the influence of the 2P on methylation of 2P-containing strands of the DNA duplexes (Table 2). M.EcoRII binds to the substrate analogue II with the same affinity as to the parent duplex I (Table 3). Replace- ment of the outer C (duplexes II and IIm) by 2P does not affect methylation of either strand (II) or the 2P-containing strand (IIm) (Tables 2 and 3). According to the thermo- dynamic and conformational analysis of the 2P-containing duplexes, the substitution of 2P for C could be a good probe for DNA–protein interactions in the major groove of DNA. Substitution of 2P for C appears to cause a small destabilizing effect and duplex II is conformationally similar to duplex I. The explanation of the observed equal binding affinities and methylation rates can be attributed to the difference in the chemical structure between 2P and C. Therefore, the 4-NH 2 group of the outer cytosine residue in the recognition sequence is not likely to be essential for sequence-specific DNA binding by M.EcoRII. In contrast, 2P substitution for both nontarget cytosine residues in the recognition sequence of MspI and HpaII C5 MTases prevents these MTases from binding, and the C4 amino functional groups of the nontarget cytosine residues are essential for DNA binding by these MTases [25]. M.EcoRII binds to duplex III containing 2P in place of the target cytosine with a K app d similar to that of duplex I (Table 3). It could be that the 4-NH 2 group of the target cytosine is not essential for recognition of DNA by M.EcoRII as was suggested for several other MTases, as base flipping probably occurs with any base at the target position [26]. However, in the case of the M.EcoRII complex with duplex III such a simple conclusion is ambiguous because in addition to the noncovalent complex a stable Fig. 3. Scatchard plot of the ratio of bound to free DNA substrate vs. concentration of bound DNA substrate. Inset: autoradiograph of gel shift assay of M.EcoRII with DNA substrate I¢. Lanes 1–4: 20 n M M.EcoRII with 1 m M AdoHcy and increasing concentrations of duplex I¢ (1,2,3.5and10n M ). Table 3. Binding and substrate properties of 2P-containing DNA duplexes. Apparent dissociation constants (K app d ) of complex M.EcoRII–DNA– AdoHcy were calculated as described in Materials and methods. Relative K app d [K app d (rel.)] were calculated as ratio of K app d of duplexes II–IV to K app d of duplex I. Relative initial methylation rates (V rel 0 ) were calculated as ratio of V 0 of duplexes II–IV to V 0 of duplex I. Recognition sequence is in bold; 2P is underlined. No DNA duplex K app d (n M ) K app d (rel.) (%) V 0 (n M Æmin )1 ) V rel 0 (%) I 5¢-GCCAACCTGGCTCT-3¢/ 4.9 ± 1.8 100 ± 37 195.6 ± 39.1 100 ± 20 3¢-CGGTTGGACCGAGA-5¢ II 5¢-GCCAA 2P CTGGCTCT-3¢/ 3.9 ± 1.4 80 ± 28 170.2 ± 56.7 87 ± 29 3¢-CGGTT-GGACCGAGA-5¢ III 5¢-GCCAAC 2P TGGCTCT-3¢/ 5.3 ± 2.4 108 ± 49 3.5 ± 0.3 1.8 ± 0.1 3¢-CGGTTG-GACCGAGA-5¢ IV 5¢-GCCAACC 2P GGCTCT-3¢/ 96.0 ± 35.6 1959 ± 726 41.1 ± 11.7 21 ± 6 3¢-CGGTTGG-ACCGAGA-5¢ Ó FEBS 2004 2-Pyrimidinone as a probe for DNA methyltransferase (Eur. J. Biochem. 271) 2395 covalent M.EcoRII complex with duplex III can be formed (see below). Thus, the K app d obtained does not represent true binding affinity of M.EcoRII to duplex III. In the case of duplexes III and IIIm, methylation was essentially not detected under steady-state conditions. The transfer of a methyl group is blocked or occurs at very low levels. In the case of duplex IV containing 2P in place of the central T in the recognition sequence, we observed a substantial increase in K app d (Table 3). Initial methylation rates of both strands (IV) or 2P-containing strand (IVm) were decreased (Tables 2 and 3). It is probable that the decrease in V 0 is attributed to the weak binding affinity of these substrate analogues to the enzyme. Recently, it was suggested that AT vs. GC discrimination is achieved by interactions between the large domain of M.EcoRII and the minor groove of DNA [13]. M.EcoRII did not bind to a substrate analogue with 2-aminopurine having been substi- tuted for adenine (M. G. Brevnov, O. A. Rechkoblit and E. S. Gromova, unpublished results). Hence, the appear- ance of the amino group in the minor groove in the case of 2-aminopurine for adenine substitution, disturbs the recog- nition of the specific DNA sequence by M.EcoRII. This is in agreement with the role of the minor groove in substrate recognition by M.EcoRII [13]. In the case of a substitution of T by 2P (duplex IV), the pattern of functional groups exposed into the minor groove remains the same, with the groups of the central thymine residue exposed into the major groove being disturbed. Therefore, it is likely that weak binding of duplex IV to M.EcoRII may be attributed to the elimination of some DNA–protein contacts in the major groove of the double helix. Alternatively, this effect may be caused by a greater destabilization of duplex IV in comparison with duplexes II and III (Table 1). However, the conformations of duplexes I–IV are similar. It has also been shown that substitution of AT by CI in the 5¢…GGT/ ACC…3¢ sequence for SinI C5 MTase led to a considerable increase in K m [13]. This observation corresponds to our suggestion that specific contacts of C5 MTases with the central base pair could be mediated by contacts not only in the minor but also in the major groove. Comparison of methylation of unmethylated and hemi- methylated DNA duplexes (Tables 2 and 3) permits us to speculate about influence of 2P on methylation of unmodi- fied DNA strand in duplexes II–IV. Equal methylation rates of duplexes II and IIm allow us to suggest that rates of methylation of unmodified and 2P-containing strands in duplex II are virtually the same. Analogously, we suppose equal methylation rates of unmodified and 2P-containing strands in duplex IV. The unmodified strand in duplex III was not methylated under steady-state conditions – prob- ably due to formation of the stable covalent adduct of M.EcoRII with 2P-containing strand. Mechanism-based inhibition of M.EcoRII by 2P -containing DNA To examine the possibility of covalent adduct formation between M.EcoRII and DNA containing 2P in place of the target C in the presence of AdoMet or AdoHcy, duplex III was incubated with the enzyme. The resulting samples were analyzed under different conditions. First, the enzyme was denaturated by adding SDS to a final concentration of 1% and subjected (or not) to heating with a subsequent analysis by 8% native PAGE (Fig. 5A). Without heating in the presence of AdoHcy, a small part of the noncovalent complex remains in the case of canonical duplex I and duplex II in which the outer cytosine residue of the recognition sequence was replaced by 2P, however, this complex is absent after heating. We did not observe the formation of the covalent adduct in the case of duplex II in the presence of AdoMet. Figure 5A (lanes 4, 5 and 9, 10) demonstrates that duplex III forms in the presence or in the absence of AdoMet a covalent intermediate with M.EcoRII stable to heating at 65 °C for 5 min. Thus, 2-pyrimidinone for the target C substitution results in the inhibition of M.EcoRII. The adducts of 2P-containing duplex III with M.EcoRII obtained in the presence of AdoHcy or AdoMet are not resistant to heating in SDS solution at 90 °Cfor5 min(data not shown) or to the addition of SDS and analysis by SDS/ PAGE (Fig. 5B, lanes 6 and 10). However, we observed products moving faster than the protein and slower than the oligonucleotides. These products seem to be oligonucleo- tides generated from the duplex III–M.EcoRII adducts. The SDS gel (Laemmli) exhibits two components at different pH: an upper part at pH 6.8 (stacking gel) and a lower part at pH 8.8 (separating gel) (Fig. 5B). Due to a pH change from 6.8 to 8.8, b-elimination of the proton from the C5 position of 2P and dissociation of the covalent intermediates of M.EcoRII and duplex III take place. The appearance of the slowly moving oligonucleotides is attributed to retarda- tion of the duplex III–M.EcoRII covalent intermediates in the upper part of the gel before dissociation. It is interesting to compare the stabilities of the adducts of C5 MTases with DNA duplexes containing AzaC, FC or 2P in place of the target C in the presence of AdoMet. The adducts of M.EcoRII with AzaC DNA [5] and M.HhaI with FC DNA [27] are resistant to heating in SDS solution Fig. 4. Binding of M.EcoRII to DNA duplex I in the presence of AdoHcy. Relative amount of M.EcoRII–DNA–AdoHcy complex obtained from the gel-shift autoradiograph vs. protein concentration is plotted. M.EcoRII (1.5–94 n M ) was incubated with duplex I (15 n M )in the presence of AdoHcy (1 m M ). Inset, autoradiograph of gel-shift assay of M.EcoRII with duplex I. Lanes: 1–8, duplex I with 1 m M AdoHcy and increasing concentrations of M.EcoRII (5, 6, 7, 10, 40, 50, 62.5 and 78 n M ); 9, duplex I. 2396 O. M. Subach et al. (Eur. J. Biochem. 271) Ó FEBS 2004 and analysis by SDS/PAGE. In contrast, the covalent intermediates of M.EcoRII with 2-pyrimidinone (duplex III) dissociate upon heating in SDS solution or during analysis by SDS/PAGE, probably because of b-elimination of the proton from the C5 position of 2-pyrimidinone. Analysis of possibility of 2-pyrimidinone methylation To clarify the role of AdoMet in the formation of the covalent adduct between 2P-containing DNA and M.Eco- RII it is important to examine the possibility of a methyl group transfer to the 2P residue. 2P-modified DNA was not methylated by MspIandHhaI C5 MTases [6]. We also did not observe the methylation of duplex III under steady-state conditions (Table 3). However, the proposed mechanism of inhibition of C5 MTases by 2-pyrimidinone [6] does not contradict the transfer of a methyl group to the 2P residue. The possibility of the methylation of DNA duplexes containing 2P in place of the target cytosine (III and IIIm) by M.EcoRII was tested at different enzyme concentrations inthepresenceofAdoMetat15°C for 30 min (Fig. 6). Hemimethylated duplex IIIm was used to exclude methy- lation of the unmodified strand. Under the same conditions, methylation of canonical duplexes I and Im was performed. The increase of enzyme concentration favoured methylation of 2P-containing DNA duplexes III and IIIm (Fig. 6). No methyl transfer was detected at all enzyme concentrations in the case of duplex 5¢-GAGCCAAGCGCACTCTGA-3¢/ 3¢-CTCGGTTCGCGTGAGACT-5¢(V) lacking the EcoR- II recognition sequence (Fig. 6). There was also no methy- lation in a control sample containing the same amount of enzyme and AdoMet but no DNA. Methylation of duplex III may be due to methyl transfer to the target unmethylated cytosine residue. However, this is impossible in the case of duplex IIIm. Hence, one can suggest that a methyl group transfer occurs to the 2-pyrimidinone base. The methylation of duplex IIIm may be stopped at the stage of formation of the covalent intermediate (Fig. 1B, step1) or may proceed with dissociation of the covalent intermediate and release of the methylated 2P-containing DNA (Fig. 1B, step2). In the first case, the quantity of methyl groups incorporated into duplex IIIm should correspond to the quantity of methyl groups incorporated into canonical DNA after the first turnover of the methylation reaction. In the second case, we should observe more than one turnover of the methylation reaction for duplex IIIm. To clarify the nature of this new effect, we compared the dependence of methylation of duplexes Im and IIIm on an enzyme concentration (Fig. 6). Complete methylation of duplex Im was observed even at low enzyme concentration. M.EcoRII transfers the methyl group to unmodified DNA strand, turns Fig. 6. Dependence of methylation of unmethylated (III), hemimethyl- ated (Im and IIIm) and nonspecific (V) DNA duplexes on concentration of M.EcoRII. M.EcoRII was incubated with indicated duplexes (500 n M ) in buffer B in the presence of [CH 3 – 3 H]AdoMet (1.3 l M )at15°Cfor 30 min. Relative methylation was calculated as the ratio of radio- activities of duplexes Im, III, IIIm and V to the radioactivity of duplex I. Methylation of duplex I (not shown) was accepted as 100%. s, Canonical duplex Im; j, duplex III; d, duplex IIIm and m, duplex V. Fig. 5. Covalent adduct formation of M.EcoRII with DNA duplexes I–III in the presence of AdoMet or AdoHcy. M.EcoRII (200 n M )was incubated with indicated duplexes (100 n M ) in buffer B in the presence of AdoHcy or AdoMet (0.5 m M ). (A). Autoradiograph of 8% native PAGE.Reactionswereincubatedwith1%SDSandheatedfor5min at 65 °C prior to electrophoresis if indicated. Lanes: 1, without M.EcoRII; 2–11, with M.EcoRII. (B). Autoradiograph of 12% SDS/ PAGE (Laemmli). Reactions were incubated with 0.8% SDS prior to electrophoresis. After autoradiography gel was stained with Coomas- sie Blue G-250, protein band is indicated by arrow; Enz is for M.EcoRII. Composition of the reaction mixtures is indicated on the top of the gels; III T (lane 1) is the upper strand of the duplex III. Stacking (upper) and separating (lower) components of the gel are shown schematically. Ó FEBS 2004 2-Pyrimidinone as a probe for DNA methyltransferase (Eur. J. Biochem. 271) 2397 over several times and, as a result, methylates all target cytosine residues for 30 min. The observed level of methy- lation of duplex IIIm was low. There was a linear increase of methylation with the increase of the enzyme concentration. This effect may be due to the arrest of the reaction after one turnover. One can suggest that the stable covalent adduct between M.EcoRII and 2P residue in DNA was formed. Its amount grew with the increase of the enzyme concentration. Therefore, for duplex IIIm, inhibition of M.EcoRII by 2P- containing DNA (i.e. the covalent intermediate is formed) occurs with methyl group transfer to the C5 position of 2P, and all active enzyme molecules become covalently bound to 2P-containing DNA (Fig. 1B, step 1). In duplex III, only one strand is modified. However, the level of methylation of duplex III was unexpectedly low (Fig. 6). We suppose that formation of the stable covalent adduct with strand containing 2P prevents effective methylation of the duplex III unmodified strand. The time dependence of methyl transfer to duplexes Im and IIIm was studied (data not shown). Most of the methyl groups were transferred to duplexes Im and IIIm by M.EcoRII within 1–2.5 min. During the remainder of the time there was very little or no further methyl transfer to DNA. We suggest that duplex IIIm forms a covalent adduct with M.EcoRII within the first few minutes of the reaction. Taken together, the results obtained suggest that the mechanism of C5 MTases inhibition by 2P in the presence of AdoMet involves methyl group transfer to the C5 position of 2P. The methylation of duplex IIIm is attributed to the formation of the stable covalent intermediate (Fig. 1B, step 1). To ascertain whether other C5 MTases can methylate 2P, methyl transfer reactions by M.HhaI to hemimethylated duplexes 5¢-GAGCCAAGCGCACTCTGA-3¢(Vm)/3¢- CTCGGTTCGMGTGAGACT-5¢ or 5¢-GAGCCAA G2PGCACTCTGA-3¢(VIm)/3¢-CTCGGTTCGMGT GAGACT-5¢ were performed at different enzyme concen- trations (Fig. 7). Duplex VIm contained 2P in place of the target cytosine in the HhaI recognition sequence (GCGC). We did not observe methylation of 2P-containing duplex VIm at low enzyme concentrations as was mentioned earlier [6]. However, methylation of duplex VIm took place at increased M.HhaI concentrations. No methyl transfer was detected in the case of duplex I lacking the HhaI recognition sequence (data not shown). Therefore, as in the case of M.EcoRII inhibition of M.HhaI by 2P was accompanied by a methyl group transfer to the 2-pyrimid- inone base. Our study allows us to assume that there are two ways of formation of covalent adducts between C5 MTases and 2P-containing DNA. In the absence of AdoMet, proton transfer to the C5 position of 2-pyrimidinone occurs (Fig. 1A) [6]. In the presence of AdoMet, methyl transfer to the C5 position of 2-pyrimidinone occurs (Fig. 1B). Similar complexes of M.EcoRII with AzaC containing DNA were reported [5]. The formation of the stable covalent intermediate between M.EcoRII and 2P-contain- ing DNA in the presence of AdoMet causes the inhibition of methylation. One can suggest that the potency of 2-pyrimidinone as an inhibitor arises from the retardation of proton elimination from the covalent intermediate in the course of catalysis as a consequence of the absence of the N4 amino group in the pyrimidinone ring. In summary, our data suggest that the conformation of DNA is not markedly affected by substitution of 2P for C or T in the sequences studied. 2-Pyrimidinone signifi- cantly destabilizes the DNA double helix in the order of sequence contexts: ACCTG > A2PCTG > AC2PTG> ACC2PG. The amino group of the outer cytosine residue in the recognition sequence does not take part in the recognition of DNA by M.EcoRII. Functional groups of the central thymine exposed in the major groove are probably involved in the recognition by the enzyme. EcoRII C5 MTase is inhibited by DNA containing 2-pyrimidinone instead of the target cytosine, two types of covalent intermediates are possible depending on the presence of AdoMet or AdoHcy. Both types of adducts undergo decomposition under heating in the presence of SDS or under analysis by SDS/PAGE. The revised mechanism of inhibition of C5 MTases by 2-pyrimidinone containing DNA may be useful in the application of 2-pyrimidinone containing DNA as a MTase inhibitor. 2-pyrmidinone incorporation in DNA sequences may also serve as a specific probe for studying discrimination contacts formed by proteins and functional groups of pyrimidine bases exposed in the major groove of DNA. Acknowledgements The research was supported by a US Public Health Service grant from the Fogarty International Center (No. TW05689) grants from the Russian Foundation for Basic Research (01-04-48637, 01-04- 48561, 02-04-48790 and 02-04-06804). We thank S. N. Mikhailov for preparation of 2-pyrimidinone phosphoramidite, S. Mu ¨ ller for oligo- nucleotide synthesis, S. Klimas ˇ auskas for M.HhaI, A. S. Bhagwat for plasmid pT71 used for construction of a hybrid plasmid carrying the gene for M.EcoRII and O. V. Kirsanova for help on purification of M.EcoRII. We are grateful to N. E. Geacintov, C. Crean and A. Kolbanovskiy for critically reading the manuscript and to V. L. Florentiev for helpful discussion. Fig. 7. Dependence of methylation of hemimethylated DNA duplex VIm on concentration of M.HhaI. M.HhaI was incubated with indicated duplex (670 n M ) in buffer B in the presence of [CH 3 – 3 H]AdoMet (420 n M )at20°C for 30 min. Relative methylation was calculated as the ratio of radioactivity of duplex VIm to the radioactivity of duplex Vm. Methylation of duplex Vm (not shown) was accepted as 100%. 2398 O. M. Subach et al. (Eur. J. Biochem. 271) Ó FEBS 2004 References 1. 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