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: or

(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. The reaction was incubated for 12 h
to run to completion and different amounts of the methy-
lated H3 methylation reaction mixtures were separated on a
15% SDS ⁄ PAGE gel together with the Dnmt3a-C after

overnight incubation with labeled AdoMet. The amount
of radioactivity incorporated in the protein bands was
determined from scanned autoradiography pictures by
densitometry.
Acknowledgement
This work was supported by DFG (JE 252-6) and
DAAD.
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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.
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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