Báo cáo khoa học: Auto-methylation of the mouse DNA-(cytosine C5)-methyltransferase Dnmt3a at its active site cysteine residue pot

Auto-methylation of the mouse DNA-(cytosine C5)- methyltransferase Dnmt3a at its active site cysteine residue Abu Nasar Siddique, Renata Z. Jurkowska, Tomasz P. Jurkowski and Albert Jeltsch Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Germany Introduction Methylation of biomolecules including proteins, DNA, RNA and small molecules plays important and diverse roles in biology [1]. For these reactions, S-adenosyl-l- methionine (AdoMet) is by far the most commonly used methyl group donor. It contains the methyl group bound to a positively charged sulfonium center; conse- quently the methyl group is highly activated towards a nucleophilic attack and AdoMet is a highly reactive compound with high methylation capacity. Overall, following ATP, AdoMet is the second most commonly used coenzyme in nature [2] and it has been estimated that about 3% of all enzymes listed in the EC nomen- clature represent AdoMet-dependent methyltransferases [3]. Methylation substrates range in size from small compounds like catechol to biopolymers like proteins, RNA and DNA; the target atoms for methylation can be carbon, oxygen, nitrogen, sulfur or even halides [4]. DNA methylation is common to almost all living organisms. In bacteria, three kinds of methylated bases are present, 5-methylcytosine, 4-methylcytosine and 6-methyladenine, whereas only 5-methylcytosine is found in higher eukaryotes [5]. In mammals, DNA Keywords auto-methylation; DNA methyltransferase; enzyme mechanism; enzyme regulation; protein methylation Correspondence T. P. Jurkowski or A. Jeltsch, Biochemistry Laboratory, School of Engineering and Science, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany Fax: +49 421 200 3249 Tel: +49 421 200 3247 E-mail: t.jurkowski@jacobs-university.de or a.jeltsch@jacobs-university.de (Received 16 December 2010, revised 28 March 2011, accepted 6 April 2011) doi:10.1111/j.1742-4658.2011.08121.x The Dnmt3a DNA methyltransferase is responsible for establishing DNA methylation patterns during mammalian development. We show here that the mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl- L-methionine (AdoMet) to a cysteine residue in its catalytic center. This reaction is irreversible and relatively slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a and enhances its AdoMet binding. Auto-methyl- ation was observed in binary Dnmt3a AdoMet complexes. In the presence of CpG containing dsDNA, which is the natural substrate for Dnmt3a, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. Therefore, this reaction might constitute a regulatory mechanism which could inactivate unused DNA methyltransferases in the cell, or it could simply be an aberrant side reaction caused by the high methyl group transfer potential of AdoMet. Enzymes Dnmt3a is a DNA-(cytosine C5)-methyltransferase, EC 2.1.1.37. Structured digital abstract l Dnmt3a methylates Dnmt3a by methyltransferase assay (View interaction) l Dnmt3a and DNMT3L methylate Dnmt3a by methyltransferase assay (View interaction) Abbreviations AdoHcy, S-adenosyl- L-homocysteine; AdoMet, S-adenosyl-L-methionine; DNA methyltransferase, MTase. FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS 2055 methylation is restricted mostly to CpG dinucleotides. The cell and tissue specific DNA methylation pattern is set early during embryonic development by the action of the Dnmt3a and Dnmt3b de novo DNA methyltransferases (MTases). Once established, the methylation pattern is further maintained during each DNA replication and cell division by the maintenance MTase Dnmt1 [6,7]. DNA methylation contributes to major biological processes, like epigenetic regulation of gene expression, genomic imprinting, X-chromosome inactivation, protection against selfish genomic ele- ments and maintenance of genomic stability [6,7]. The Dnmt3a MTase comprises a large N-terminal regulatory domain and a C-terminal catalytic domain, which is active in an isolated form [8]. The catalytic domain shares a well conserved structure with all DNA MTases, called ‘AdoMet-dependent MTase fold’, which consists of a mixed seven-stranded b-sheet, formed by six parallel and the seventh anti-parallel b strands, inserted between the fifth and sixth b strands. This central b-sheet is sandwiched between six a-helices [9,10]. Because the target base is buried in the DNA helix and not readily accessible for catalysis, DNA MTases flip out the target base and insert it in a hydrophobic pocket in their active center [11]. The catalytic mechanism used by Dnmt3a is characteristic for the DNA-(cytosine C5)-MTases and it is mainly involved in activation of the substrate by increasing its nucleophilicity [5]. For that purpose, DNA-(cytosine C5)-MTases use a catalytic cysteine residue to perform a nucleophilic attack on the sixth position of the cyto- sine, which leads to the formation of a covalent bond between the enzyme and the substrate base. The for- mation of the cysteine–cytosine bond increases the neg- ative charge density at the C5 atom of the cytosine, which then attacks the methyl group bound to Ado- Met. Base flipping and the nucleophilic attack of the cysteine are facilitated by a contact of a conserved glu- tamate residue to the exocyclic amino group and the N3 ring nitrogen atom. In addition, a conserved argi- nine residue plays a role in base flipping and catalysis. Exchange of any of these residues leads to a reduction or complete loss of the catalytic activity of Dnmt3a [12,13]. Results Detection of auto-methylation of Dnmt3a-C The methylation of lysine and arginine residues of histones is an important post-translation modification involved in regulation of gene expression and chroma- tin biology [6,14,15]. However, recently the regulatory function of lysine methylation of non-histone proteins has moved into the focus of research [16,17]. To look into the possible regulation of Dnmt3a-C by lysine methylation, we investigated the potential lysine meth- ylation of the Dnmt3a-C enzyme by several mamma- lian protein lysine methyltransferases. To this end, purified Dnmt3a-C was incubated with different protein lysine methyltransferases in the presence of S-[methyl-3H]-adenosyl-l-methionine (AdoMet) with radioactively labeled methyl group in order to detect the transfer of radioactivity to Dnmt3a-C. Afterwards samples were analyzed by SDS ⁄ PAGE electrophoresis and autoradiography. However, after incubation of Dnmt3a-C with radioactively labeled AdoMet for longer periods of time, we detected the transfer of radioactivity to the Dnmt3a-C protein even without addition of a protein methyltransferase (Fig. 1). This modification was resistant to heat (95 °C for 5 min in the presence of 2% SDS and 5 mm dithiothreitol); therefore, it seemed to be of covalent nature and it most probably resulted from an intrinsic auto-methyla- tion activity of the enzyme. A similar observation was also made with full-length Dnmt3a2 (Fig. 1C), which is the predominant isoform of Dnmt3a in embryonic stem cells and embryonal carcinoma cells [18]. Since we suspected this covalent labeling of Dnmt3a-C would inhibit the enzyme and it could have a regulatory role, we decided to study the phenomenon in more detail. Literature searches uncovered similar observations already made for some bacterial MTases, including the DNA-(cytosine C5)-MTases M.BspRI [19,20] and Dcm [21] and the DNA-(adenine N6)- MTase M.EcoPI [22]. For M.BspRI, it was suggested that the methyl group can be directly transferred from the AdoMet to a cysteine residue of the protein, lead- ing to the formation of a chemically stable S-methylcy- steine and resulting in inactivation of the enzyme [19]. Kinetics and irreversible nature of the auto-methylation To follow the time course of auto-methylation, we have incubated Dnmt3a-C with radioactively labeled AdoMet and removed aliquots from the reaction mix- ture at different time points. Reactions were stopped by the addition of SDS to a final concentration of 2mm, followed by heat denaturation of the protein and SDS ⁄ PAGE electrophoresis. The extent of radio- activity bound to the protein was visualized by autora- diography and quantified by densitometry. As shown in Fig. 1A and B, the radioactive signal is increasing slowly over the course of hours with a roughly linear increase for the first 4 h of the reaction. Fitting of the Auto-methylation of the Dnmt3a methyltransferase A. N. Siddique et al. 2056 FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS reaction progress curve to a single exponential rate equation gave an estimate of 0.1 h )1 for the auto- methylation rate constant, indicating that auto-methyl- ation is a slow process in contrast to the non-covalent AdoMet binding or exchange which happens within minutes [12]. Furthermore, non-covalently bound Ado- Met will not co-migrate with Dnmt3a-C in denaturing gel electrophoresis. Hence, we conclude that indeed an auto-methylation of Dnmt3a-C occurs. To confirm the irreversible nature of the labeling of Dnmt3a-C, reactions were quenched with unlabeled AdoMet and AdoMet analogs. An initial auto-methyl- ation reaction was performed for 1 h, allowing the for- mation of some auto-methylated Dnmt3a-C, and then a large excess of unlabeled AdoMet or S-adenosyl-l- homocysteine (AdoHcy) was added to the reaction and samples were taken at 1 h intervals and analyzed by autoradiography. As expected, the addition of either unlabeled AdoMet or AdoHcy inhibited the further incorporation of radioactivity into the Dnmt3a-C pro- tein (Fig. 2A). However, already incorporated radioac- tivity remained, indicating that the modification is stable and irreversible under in vitro conditions. Auto-methylation occurs at the catalytic cysteine Taking into account the catalytic mechanism of Dnmt3a-C, it seemed very likely that the catalytic cys- teine residue was the methyl group acceptor because it lies in close proximity to the methyl group of AdoMet and is the most reactive residue in the catalytic center of the enzyme. To test whether the catalytic cysteine is the target for auto-methylation, we purified the alanine exchange mutant C120A of Dnmt3a-C [13] and incu- bated it with radioactively labeled AdoMet. As expected, the C120A mutant Dnmt3a did not get labeled (Fig. 2B), strongly suggesting that the active site cysteine is the target of modification. Coomassie kDa 0‘ 5‘ 15‘ 30‘ 1 h 2 h 4 h 14 h Autoradiography BA 15 25 35 40 55 70 100 130 170 25 35 40 Time C kDa 25 35 40 55 70 100 130 170 kDa 25 35 40 55 70 100 130 170 Autoradiography Coomassie 0 3.0E + 06 2.5E + 06 2.0E + 06 1.5E + 06 1.0E + 06 5.0E + 05 0.0E + 00 5 Time (h) Rel. intensity (a.u.) 10 15 Lane 1 Lane 2 Lane 3 Lane 1 Lane 2 Lane 3 Fig. 1. Auto-methylation of the Dnmt3-C DNA methyltransferase. (A) Dnmt3-C protein was incubated with radioactively labeled AdoMet in the standard reaction buffer for the indicated time periods. Reactions were stopped and samples split into equal parts and both run on 15% SDS ⁄ PAGE. The first gel was fixed, sensitized, dried, and then exposed to X-ray film for 5 days (labeled Autoradiography). The second gel was stained with colloidal Coomassie to serve as loading control (labeled Coomassie). Dnmt3a-C migrates in the gel with an apparent mass of  37 kDa. (B) Quantification of the autoradiography signal coming from the Dnmt3a-C protein band. The exposed and developed X-ray films were scanned and the strength of the radioactivity signal was estimated using densitometry; the intensity values (a.u.) were plotted as a function of time and fitted to a single exponential rate equation. (C) Auto-methylation of full-length Dnmt3a. The methylation was performed for 14 h. Nine, 6 or 3 lg of the protein were loaded on polyacrylamide gels (lanes 1–3) and subjected to autoradiography or Coomassie staining. A. N. Siddique et al. Auto-methylation of the Dnmt3a methyltransferase FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS 2057 15 25 35 40 55 70 100 130 170 1 h2 h5 h2 h5 ho/n2 h5 ho/n Dnmt3a-C + 3 H-AdoMet Dnmt3a-C + 3 H-AdoMet + AdoMet Dnmt3a-C + 3 H-AdoMet + AdoHcy 3 h o/n 3 h 3 h0 h Autoradiography Coomassie MW 0 h 2 h 4 h o/n 0 h 2 h 4 h o/n 0 h 2 h 4 h o/n 0 h 2 h 4 h o/n Dnmt3a-C C120A + 3 H-AdoMet Dnmt3a-C + 3 H-AdoMet 15 25 35 40 55 70 100 130 170 Dnmt3a-C C120A + 3 H-AdoMet Dnmt3a-C + 3 H-AdoMet kDa kDa 15 25 35 40 55 70 100 130 170 Autoradiography + AdoMet + AdoHcy B A kDa C Dnmta-C wt Dnmta-C wt + AdoMet 5000 4000 3000 2000 1000 0 2500 2000 1500 1000 500 0 m/z Intensity (a.u.) 13.96 2934.347 2948.344 13.96 2920 2925 2948.303 2934.347 2930 2935 2940 2945 2950 2955 2960 2965 Fig. 2. The Dnmt3a auto-methylation reaction is irreversible, dependent on AdoMet and occurs at the catalytic cysteine residue. (A) An auto- methylation reaction was incubated for 1 h to allow for creation of auto-methylated species. Then the reaction was quenched by addition of 1000-fold molar excess of either unlabeled AdoMet or AdoHcy. For reference the auto-methylation reaction was continued without addition of a quencher. Aliquots from the reactions were taken at 2 h, 3 h, 5 h and 12–14 h after the addition of quencher and run on a 15% SDS ⁄ PAGE; the amount of incorporated 3H-methyl groups was checked by autoradiography. (B) Purified C120A and wild-type Dnmt3a-C were incubated with 3 H-AdoMet for 0 h, 2 h, 4 h and 12–14 h in the reaction buffer. Two aliquots from the reaction were taken at each time point and run sep- arately on two 15% SDS ⁄ PAGE gels, from which one was stained with colloidal Coomassie G-250 and served as loading control and the other was used for autoradiography. (C) Mass spectroscopic analysis of auto-methylation of Dnmt3a-C with and without incubation with unlabeled AdoMet (1 m M). The tryptic fragment containing the active site Cys120 has a mass of 2934.3 Da (theoretical mass 2934.4 Da). After incubation with AdoMet an additional peak appears at 2948.3 Da corresponding to 2934.3 Da plus the mass of a methyl group (14 Da). Auto-methylation of the Dnmt3a methyltransferase A. N. Siddique et al. 2058 FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS To confirm that auto-methylation occurs at Cys120, Dnmt3a-C was incubated with unlabeled AdoMet and subjected to tryptic digestion and MALDI-TOF mass spectrometric analysis. As shown in Fig. 2C, the peak corresponding to the unmethylated peptide containing Cys120 as well as the peak corresponding to the methylated peptide were clearly detectable. This tryptic fragment did not contain another amino acid residue that could function as nucelophile (Fig. S1). The methylated peak was not detected with a control sam- ple that was not pre-incubated with AdoMet (Fig. 2C). As an additional control, the C120A variant was incu- bated with AdoMet and subjected to mass spectromet- ric analysis. In this experiment the peptide containing the C120A mutation was detectable, but neither a peak corresponding to the methylated C120A peptide nor a peak corresponding to the methylated C120 peptide was observed (Fig. S2). Our identification of the active site cysteine residue as the target for auto-methylation parallels literature findings with other enzymes. In the case of M.BspRI, Szilak and colleagues have identified two cysteine resi- dues which were the targets for auto-methylation: one of them (C156) is the catalytic cysteine in M.BspRI; the other one (C181) is not conserved among DNA- (cytosine-C5)-MTases [19]. In the case of Dcm only the catalytic cysteine residue was found to get modified [21]. Extent of auto-methylation In order to estimate the fraction of Dnmt3a-C which gets self-methylated, we compared the radioactivity sig- nal generated by Dnmt3a-C after 16 h incubation under our standard reaction conditions with the signal coming from histone H3.1 monomethylated at lysine 4 by recombinant SET7 ⁄ 9 histone lysine MTase [23–25]. As shown in Fig. 3, the autoradiography signal of the Dnmt3a-C protein is faint in comparison with the sig- nal of histone H3. Taking into consideration the rela- tive strength of the autoradiography signals and the total protein amounts of Dnmt3a-C and H3.1 loaded on the gel, we estimated that about 2.6% of the Dnmt3a-C got modified during this 16 h incubation. It is interesting that the extent of auto-methylation observed in the mass spectrometric analysis (Fig. 2C) was much higher than the extent of methylation observed in Fig. 3. Although this observation needs to be interpreted carefully, since mass spectroscopy is not a fully quantitative method, the higher methylation may be related to the fact that the concentration of AdoMet was much higher in the mass spectrometric experiment (1 mm in the assay) than in the methyla- tion with radioactively labeled AdoMet (0.76 lm in the assay). Effect of DNA and Dnmt3L on auto-methylation of Dnmt3a It is known that Dnmt3L, an activator of Dnmt3a and Dnmt3b, stimulates the DNA methylation reaction catalyzed by these enzymes [26]. As shown in Fig.4, larger amounts of radioactivity were transferred to the Dnmt3a-C protein after adding Dnmt3L-C to the auto-methylation reaction compared with the reaction mixture with Dnmt3a alone. This result can be explained because Dnmt3L stabilizes the conformation of the active site loop of Dnmt3a-C and it increases AdoMet binding [9,26], which in turn will lead to increased formation of self-methylated Dnmt3a-C. SET7/9 Histone H3.1 Dnmt3a-C 10 15 25 35 40 55 70 100 130 170 156 SET7/9 + histone H3.1 D3a-C 2.6 6.5 13 26 52 pmol of protein kDa BA Autoradiography Fig. 3. Quantification of the extent of auto-methylation. (A) Comparison of the radioactivity signal from auto-methylated Dnmt3a-C (labeled D3a-C) after overnight (14 h) incubation with 3H-AdoMet in the standard reaction conditions with signal from different amounts of SET7 ⁄ 9 methylated histone H3.1 (NEB). The total protein amounts loaded on the gel are indicated. (B) The band intensities from the autoradiography picture were extracted by densitometry and background normalized. A. N. Siddique et al. Auto-methylation of the Dnmt3a methyltransferase FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS 2059 To test the effect of DNA on the auto-methylation reaction of Dnmt3a-C, we added a 20 bp double- stranded DNA containing a single CG target site and followed the incorporation of radioactivity from Ado- Met into DNA and Dnmt3a-C. As can be seen in Fig. 4, addition of a double-stranded DNA substrate abolished the tritium incorporation into the MTase but at the same time the DNA got efficiently methylat- ed. This result illustrates that after binding both sub- strates (DNA and AdoMet) the enzyme has a high specificity for the transfer of the methyl group to the DNA and it efficiently avoids auto-methylation. Discussion We show here that the mammalian Dnmt3a enzyme undergoes auto-methylation in vitro at its catalytic cys- teine by transferring the methyl group from its natural cofactor AdoMet to the cysteine residue. Analogous reactions were already observed for the bacterial DNA-(adenine N6)-MTase M.EcoPI [22] and the two DNA-(cytosine C5)-MTases Dcm [21] and M.BsuRI [19,20], but not for a mammalian DNA MTase. This observation highlights one interesting detail in the cat- alytic mechanism of DNA-(cytosine C5)-MTases. On one hand, these enzymes employ AdoMet as the donor for methyl groups, which is a highly activated coen- zyme with very large methyl group transfer potential. On the other hand, they harbor a cysteine residue in their active centers that is activated towards perform- ing a nucleophilic attack. This cysteine residue could easily react with AdoMet because the DG° for the transfer of the methyl group from AdoMet to cysteine is of the order of )70 kJÆmol )1 [2,4]. Therefore, it is essential for the enzyme that a close approximation of these two groups is avoided to prevent auto-methyla- tion and inactivation of the enzyme. Indeed, in the Dnmt3a-C structure with AdoHcy, the sulfhydryl and AdoHcy sulfur atoms are separated by 7.66 A ˚ , which would correspond to a distance of about 6 A ˚ between the sulfhydryl sulfur and the methyl group of AdoMet, if AdoMet were to replace AdoHcy without conforma- tional change (Fig. 5). This suggests that a confor- mational change of about 3 A ˚ has to occur before auto-methylation can happen, which may explain why the process of auto-methylation is slow. We show here that the conformation of Dnmt3a prevents auto-meth- ylation efficiently but not entirely, similar to what has been observed with the bacterial enzymes mentioned above. However, if DNA is bound, no auto-methyla- tion is happening, suggesting that the reaction occurs in binary Dnmt3a-C AdoMet complexes but in ternary 10 15 25 35 40 55 70 100 130 170 2 h 6 h 12 h 2 h 6 h 12 h 2 h 6 h 12 h Dnmt3a-C Dnmt3a-C + Dnmt3L Dnmt3a-C + CG DNA Dnmt3a-C DNA kDa Autoradiography Fig. 4. Effect of Dnmt3L and DNA on the auto-methylation reac- tion. Comparison of efficiencies of auto-methylation reactions car- ried out in the presence of 7 l M Dnmt3L C-terminal domain or 20 l M CG containing dsDNA with the standard auto-methylation reaction. Whereas Dnmt3L increases the level of auto-methylation of Dnmt3a-C, the methylation of the dsDNA substrate is efficiently competing with the Dnmt3a-C auto-methylation. Note that oligonu- cleotides are not fully denatured in SDS gels and run as a mixture of single-stranded and double-stranded form. In addition, binding of Dnmt3a-C to the DNA causes the appearance of an additional retarded oligonucleotide band. 7.66 Å Fig. 5. Positioning of AdoHcy and the catalytic cysteine in the active center of Dnmt3a-C, in the crystal structure of Dnmt3a ⁄ Dnmt3L complex with AdoHcy [9] (PDB: 2QRV). AdoHcy is shown in orange, Dnmt3a colored by atom type. The distance between the sulfhydryl atom of the catalytic cysteine side chain and sulfur atom of AdoHcy (7.66 A ˚ ) is indicated. Auto-methylation of the Dnmt3a methyltransferase A. N. Siddique et al. 2060 FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS complexes the transfer of the methyl group to the flipped target base is much preferred. It is unclear whether this slow auto-methylation of Dnmt3a could have a biological function in cells. Since the methylcysteine is chemically stable, auto-methyla- tion would inactivate the enzyme by causing steric con- straints and interference with the reaction mechanism of DNA-(cytosine C5)-MTases. It is possible that, in cells, Dnmt3a which is in an idle state may lose its activity via auto-methylation, thereby protecting the genome against aberrant methylation. In this respect it is important to note that we observed much higher lev- els of auto-methylation after incubation of the Dnmt3a-C at higher concentration of AdoMet. The intracellular concentrations of AdoMet have been reported in the range of 50–250 lm [27–30], which is much higher than the AdoMet concentration used here in the radioactive methylation (0.76 lm) and may allow for more efficient auto-methylation in the cell. There also might be additional factors in the cells, which could stimulate Dnmt3a for auto-methylation and therefore inactivation. On the other hand, the auto-methylation of Dnmt3a and other MTases may simply be a side reaction caused by the high methyl group transfer potential of AdoMet. Experimental procedures The His 6 -tagged full-length Dnmt3a2 and the catalytic domain of mouse Dnmt3a wild-type and catalytically inac- tive C120A mutant (corresponding to C706A in full-length Dnmt3a) and the His 6 -tagged fusion of the C-terminal part of human DNMT3L were expressed and purified as described previously [26,31]. The human SET7 ⁄ 9 protein lysine methyltransferase which among other sites monome- thylates histone H3 at Lys4 [24,25] was purified as described earlier [32]. Auto-methylation reaction mixtures contained 2–5 lm Dnmt3a-C protein, 0.76 lm [methyl- 3 H]-AdoMet (PerkinEl- mer) in methylation buffer (20 mm Hepes pH 7.2, 1 mm EDTA, 50 mm KCl) in a 20 lL reaction volume. Reactions were incubated at room temperature for various time inter- vals and stopped by addition of 20 lL of Laemmli sample buffer (130 mm Tris ⁄ HCl pH 6.8, 20% glycerol, 4% SDS, 10 mm dithiothreitol, 0.02% bromophenol blue) and heat- ing to 95 °C for 5 min. Afterwards, the samples were ana- lyzed on a 15% SDS ⁄ PAGE gel and either stained with colloidal Coomassie (33) or fixed with 10% methanol ⁄ 10% acetic acid, immersed in Amplify solution (GE Healthcare, Freiburg, Germany) for 1 h at room temperature with shaking, dried on a 3 mm Whatman paper and exposed to an X-ray film for 3–7 days. Signal intensities were analyzed by densitometry (AIDA v4; raytest GmbH, Straubenhardt, Germany). Auto-methylation reactions with unlabeled Ado- Met were carried out in the same buffer but in the presence of 1 mm AdoMet (Sigma-Aldrich) for 16 h. Unlabeled AdoMet was dissolved in 10 mm sulfuric acid, stored in aliquots at )20 °C and used only once after thawing. Quenching reactions were prepared essentially like auto- methylation reactions; however, the auto-methylation reac- tions were incubated for 1 h allowing the formation of some initial auto-methylated species and then quenched by the addition of either 1 mm non-radioactive AdoMet (Sigma-Aldrich), 1 mm AdoHcy (Sigma-Aldrich) or 20 lm of double-stranded CG-DNA (GAA GCT GGG ACT TC C GGA GGA GAG TGC AA). The samples were col- lected at various time points and analyzed as described above. To study the effect of Dnmt3L on the auto-methyla- tion reaction of Dnmt3a-C, auto-methylation reactions were supplemented with 7 l m recombinant Dnmt3L and analyzed as described above. To calibrate the extent of auto-methylation of Dnmt3a, human recombinant histone H3.1 (NEB) was methylated with 1.22 lm recombinant SET7 ⁄ 9. H3 methylation was performed in methylation buffer for SET7 ⁄ 9 (50 mm Tris ⁄ HCl pH 9.0, 5 mm MgCl 2 ,4mm dithiothreitol) using 0.76 lm 3H-AdoMet. 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N. Siddique et al. 2062 FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS 33 Dyballa N & Metzger S (2009) Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. J Vis Exp 30, 1431. Supporting information The following supplementary material is available: Fig. S1. Sequence of the tryptic peptide containing Cys120 and its position in the Dnmt3a structure. Fig. S2. Absence of auto-methylation w ith the Dnmt3a-C C120A variant. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. A. N. Siddique et al. Auto-methylation of the Dnmt3a methyltransferase FEBS Journal 278 (2011) 2055–2063 ª 2011 The Authors Journal compilation ª 2011 FEBS 2063 . loss of the catalytic activity of Dnmt3a [12,13]. Results Detection of auto-methylation of Dnmt3a- C The methylation of lysine and arginine residues of histones. in the cells, which could stimulate Dnmt3a for auto-methylation and therefore inactivation. On the other hand, the auto-methylation of Dnmt3a and other