Dnmt3a and Dnmt1 functionally cooperate during
de novo
methylation of DNA
Mehrnaz Fatemi*, Andrea Hermann*, Humaira Gowher and Albert Jeltsch
Institut fu
¨
r Biochemie, Justus-Liebig-Universita
¨
t, Gießen, Germany
Dnmt3a is a de novo DNA methyltransferase that modifies
unmethylated DNA. In contrast Dnmt1 shows high pre-
ference for hemimethylated DNA. However, Dnmt1 can be
activated for the methylation of unmodified DNA. We show
here that the Dnmt3a and Dnmt1 DNA methyltransferases
functionally cooperate in de novo methylation of DNA,
because a fivefold stimulation of methylation activity is
observed if both enzymes are present. Stimulation is obser-
ved if Dnmt3a is used before Dnmt1, but not if incubation
with Dnmt1 precedes Dnmt3a, demonstrating that methy-
lation of the DNA by Dnmt3a stimulates Dnmt1 and that
no physical interaction of Dnmt1 and Dnmt3a is required. If
Dnmt1 and Dnmt3a were incubated together a slightly increa-
sed stimulation is observed that could be due to a direct
interaction of these enzymes. In addition, we show that
Dnmt1 is stimulated for methylation of unmodified DNA if
the DNA already carries some methyl groups. We conclude
that after initiation of de novo methylation of DNA by
Dnmt3a, Dnmt1 becomes activated by the pre-existing
methyl groups and further methylates the DNA. Our data
suggest that Dnmt1 also has a role in de novo methylation of
DNA. This model agrees with the biochemical properties of

process (together with specific demethylation events) cre-
ates the pattern of DNA and therefore transfers the
epigenetic information to the DNA.
DNA methylation is introduced by DNA methyltrans-
ferases (MTases) which use S-adenosylmethionine as donor
for an activated methyl group [reviews 3,14,15,16]. Four
candidate DNA MTases have been identified in mammals
so far: Dnmt1, Dnmt2, Dnmt3a and Dnmt3b. Results
obtained with Dnmt1 knock-out mice have implicated this
enzyme in maintenance methylation [6], a role that is in
agreement to its pronounced preference for methylation of
hemimethylated DNA in vitro [17,18]. However, Dnmt1
also shows capabilities for de novo methylation of DNA
[15]. Interestingly, the de novo activity of Dnmt1 is
stimulated by binding of methylated DNA to an allosteric
site located in the N-terminal domain of the enzyme
[18,19]. However, de novo methylation activity is also
associated to the Dnmt3a and Dnmt3b enzymes, which
do not show a preference for methylation of hemimeth-
ylated CG sites [20–23]. It has been shown that one target
for the Dnmt3b enzyme are satellite sequences [7,24,25],
whereas specific targets for the Dnmt3a enzyme are not
yet known. In biochemical studies, it has been shown that
Dnmt3a methylates DNA in a distributive mechanism
[21]. This was a surprising observation, because it makes
the enzyme badly adapted for a fast methylation of one
domain of the DNA. However, the intriguing possibility
appeared that Dnmt3a and Dnmt1 might functionally
cooperate during de novo methylation of DNA. This
model assumes that Dnmt3a might initiate de novo

[methyl-
3
H]-S-adenosylmethionine (5.55 · 10
14
BqÆmol
)1
,
Moravek Biochemicals, Brea, CA) in methylation buffer
(20 m
M
Hepes pH 7.5, 50 m
M
KCl, 50 lgÆmL
)1
BSA).
Premethylation of the 310mer PCR fragment (6 pmol) was
performed with 12 U M.HpaII (NEB) for 2 h at 37 °C. The
DNA was then precipitated and the concentration deter-
mined. To ensure complete methylation of all HpaII sites in
this experiment an aliquot of the purified methylated DNA
was subjected to a second round of methylation with
M.HpaII using radiolabeled AdoMet. No radioactivity was
incorporated into the DNA demonstrating that all HpaII
sites are fully methylated.
RESULTS AND DISCUSSION
It was the aim of this work to experimentally investigate a
model for de novo methylation of DNA that postulates
functional cooperation of Dnmt1 and Dnmt3a. Purified
Dnmt1 and Dnmt3a enzymes were employed to methylate a
958mer PCR product using labeled [methyl-

we have also carried out reactions in which incubation of the
DNA with Dnmt1 was followed by incubation with
Dnmt3a or in which the DNA was incubated twice with
Dnmt3a. As shown in Fig. 2 incubation of Dnmt3a before
Dnmt1 but not Dnmt1 before Dnmt3a results in stimulation
of the DNA methylation activity. This finding supports the
Fig. 1. Stimulation of DNA methylation by cooperation of Dnmt3a and
Dnmt1. A 958mer PCR product (20 n
M
) was methylated using labeled
[methyl-
3
H]-AdoMet by Dnmt1 (80 n
M
)(j), Dnmt3a (400 n
M
)(d)
and both enzymes present at the same time (r). All experiments were
carried out in triplicate. We observed a 4.7 ± 0.1-fold higher rate of
DNA methylation in the presence of Dnmt3a and Dnmt1 than the sum
of the rates observed with either enzyme alone.
Fig. 2. Stimulation of DNA methylation observed with different mixing
protocols of Dnmt1 and Dnmt3a. In the experiment labeled D3a/D3a,
methylation of 958mer (20 n
M
) was carried out with Dnmt3a (400 n
M
)
for 30 min. Then, the DNA was precipitated with ethanol and a sec-
ond methylation reaction was carried out with Dnmt3a (400 n

stimulation to occur. We repeatedly observed a slightly
higher activity if both enzymes are present at the same time.
However, this effect is only small and close to the error
margins of our experiment. This difference could be due to a
direct interaction of Dnmt1 and Dnmt3a, which has
recently been reported to occur [27].
It is known that Dnmt3a creates hemimethylated target
sites during DNA methylation [28]. As the activity of
Dnmt1 on hemimethylated DNA is much higher [18],
generation of one hemimethylated site will immediately lead
to a second methylation in the unmodified strand of this site
in the presence of Dnmt1. Kinetically this reaction scheme
means that two methyl groups are always introduced, one
slowly by Dnmt3a or by de novo methylation catalyzed by
Dnmt1 and the second one very fast. Therefore, this effect
can only double the rate of DNA methylation, because
every turnover by Dnmt3a can trigger only one turnover by
Dnmt1 in the opposite strand of the DNA. Consequently,
this effect cannot account for the fivefold stimulation
observed in our experiments and additional stimulation of
Dnmt1 by pre-existing methylation must occur. To show
this effect we used a 310mer PCR fragment that contains
three fully methylated CG sites produced by methylation
with M.HpaII. As shown in Fig. 3 the premethylated DNA
is modified significantly faster by Dnmt1 confirming similar
results by other groups [29,30]. Furthermore, it is in
agreement with the allosteric activation of Dnmt1 observed
after binding of the enzyme to methylated oligonucleotides
[18,19,31,32]. A similar effect is not observed with Dnmt3a
showing that at least under these conditions no allosteric

domain whereas without the cooperation with Dnmt1,
Dnmt3awouldhavetorebindtotheDNAaftereach
turnover to achieve complete methylation of one domain of
the DNA. It should be noticed that the molecular mechan-
ism of these targeting events is still unknown, although the
interaction of Dnmt3a with some other proteins has been
shown that might be the mechanistic basis for the process
[35]. Finally, it is possible that maintenance methylation at
sequences that are not efficiently methylated by Dnmt1
alone also follows this mechanism.
Our data contribute to the view that DNA methylation is
a complicated process, in which different enzymes are
involved: We show here that Dnmt1 and Dnmt3a can
cooperate in de novo methylation in vitro, a similar
phenomenon has also been observed in vivo [34]. These
authors also have shown that the Dnmt3a and Dnmt3b
enzymes (in addition to Dnmt1) are required for mainten-
ance of the methylation at certain DNA sequences. A role of
Fig. 3. Stimulation of Dnmt1 and Dnmt3a by fully methylated CpG sites. In this experiment the rates of methylation of the 310mer substrate were
compared after premethylation of the substrate on 3 HpaII sites (CCGG) located at the 5¢ end of the molecule (j) and without premethylation (d).
In the left panel 40 n
M
DNA were methylated using 120 n
M
Dnmt1, in the right panel using 400 n
M
Dnmt3a. In repeated experiments a 2.9 ± 0.2-
fold stimulation was observed with Dnmt1, whereas no significant difference was observed with Dnmt3a.
Ó FEBS 2002 Cooperation of Dnmt1 and Dnmt3a (Eur. J. Biochem. 269) 4983
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