From the Institut für Biochemie, Fachbereich Biologie,
Justus-Liebig-Universität, Heinrich-Buff-Ring 58, D-35392 Giessen, Germany
All DNA methyltransferases (MTases) have similar
catalytic domains containing nine blocks of conserved amino acid
residues. We have investigated by site-directed mutagenesis the
function of 17 conserved residues in the EcoRV
-adenine-N6-DNA methyltransferase.
The structure of this class of MTases has been predicted recently. The
variants were characterized with respect to their catalytic activities
and their abilities to bind to DNA and the
S-adenosylmethionine (AdoMet) cofactor. Amino acids located
in motifs X, I, and II are shown to be involved in AdoMet binding
(Lys16, Glu37, Phe39, and
Asp58). Some of the mutants defective in AdoMet binding are
also impaired in DNA binding, suggesting allosteric interactions
between the AdoMet and DNA binding site. Asp78 (motif III),
which was supposed to form a hydrogen bond to the AdoMet on the basis
of the structure predictions, turned out not to be important for AdoMet
binding, suggesting that motif III has not been identified correctly.
R128A and N130A, having mutations in the putative DNA binding domain,
are unable to bind to DNA. Residues located in motifs IV, V, VI, and
VIII are involved in catalysis (Asp193, Tyr196,
Asp211, Ser229, Trp231, and
Tyr258), some of them presumably in binding the flipped
target base, because mutations at these residues fail to significantly
interfere with DNA and AdoMet binding but strongly reduce catalysis.
Our results are in substantial agreement with the structure prediction for EcoRV
-adenine-N6-methyltransferase and
x-ray structures of other MTases.
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INTRODUCTION |
Methylation of DNA is essential in mammals. It is involved in
regulation of gene expression, imprinting, development, chromatin structure, DNA replication, and origin of cancer (for reviews, see
Refs. 1-5). DNA can be modified by two classes of DNA
methyltransferases (MTases),1
viz. C-MTases modifying cytosines at the
C5-position and N-MTases transferring a methyl
group to cytosine-N4- or
adenine-N6-positions. Prokaryotic DNA
MTases comprise approximately 250-400 amino acid residues (for
reviews, see Refs. 6-9). The C-terminal domain of eukaryotic enzymes
(for a review, see Ref. 1) bears homology to the prokaryotic
cytosine-C5-MTases, which contain 10 conserved
amino acid motifs (for reviews, see Refs. 8 and 10).
So far the structures of two prokaryotic C-MTases are known,
M. HhaI (11-14) and HaeIII DNA methyltransferase
(15). Both enzymes consist of two domains. One large, catalytic domain
forms the binding site for the cofactor S-adenosylmethionine
(AdoMet) and the catalytic center and harbors nine of the 10 conserved amino acid motifs. One smaller domain is responsible for DNA
recognition. The catalytic mechanism of these enzymes involves flipping
out the target base from the DNA helix (12). Then a conserved cysteine (motif IV) performs a nucleophilic attack on the
C6-position of the cytosine, forming a covalent enzyme-DNA
intermediate. Thereby, the C5-position is activated and
accepts the methyl group from AdoMet. Subsequently, the covalent
intermediate is deprotonated at C5 leading to the
elimination of the cysteine (16, 12, 14).
N-MTases contain nine conserved amino acid motifs. An
FXGXG motif, also present in
cytosine-C5-MTases, and a DPPY motif (consensus
sequence, (S/N/D)PP(Y/F/W)) are moderately conserved (17-20), and the
homologies of seven additional motifs are weak. Consequently, these
additional motifs only recently could be identified in structure-guided
alignments (21). Whereas some amino acid residues within the
FXGXG and DPPY motifs have already been
investigated by site-directed mutagenesis (22-25), no mutational
studies have been carried out so far to test the functional roles of
conserved amino acid residues within the other motifs either in
C- or N-MTases.
According to the amino acid sequences and order of the conserved
motifs, N-MTases can be subdivided into three distinct
groups, viz.
-N-MTases (e.g. M. EcoRV, an adenine-N6 MTase),
-N-MTases (e.g. PvuII DNA
methyltransferase, a cytosine-N4-MTase),
and
-N-MTases (e.g. M. TaqI, an
adenine-N6-MTase) (20, 21). Eukaryotic
enzymes involved in mRNA processing, double-stranded RNA adenosine
deaminase (26), and
mRNA(adenosine-N6)-methyltransferase
(27) are similar to DNA N-MTases. So far, structures are
available only for
- and
-type apoenzymes (M. TaqI
(28) and PvuII DNA methyltransferase (29)). Like
C-MTases, N-MTases are built up of two domains.
Surprisingly, the catalytic domain of N-MTases has a similar
structure as the catalytic domains of C-MTases, although the chemistry
of catalysis is different (30). Interestingly, the conserved DPPY motif
structurally corresponds to motif IV in C-MTases, which
contains the catalytic cysteine residue. It is likely that
N-MTases, like C-MTases, flip their target base
out of the DNA double helix (31-34).
The EcoRV DNA methyltransferase specifically methylates the
first adenine within the EcoRV recognition sequence GATATC
(35). The enzyme is well characterized with respect to its DNA binding and kinetic properties (33, 34, 36-39). Using multiple sequence alignments (Fig. 1) as well as secondary
structure predictions the structure of M. EcoRV and other
-type N-MTases was predicted to contain a similar catalytic domain as the other MTases (Fig. 2) (21,
40). On the basis of these predictions, putative functional roles for
several conserved amino acid residues (Fig. 1) can be derived,
e.g. involvement in DNA binding, AdoMet binding, or
catalysis (Fig. 2). In particular, a
putative binding site for the flipped target base can be derived by
comparison with C-MTases (31, 21). Here we report the
results of an extensive mutational study of 17 conserved amino acid
positions of M. EcoRV that allow us to draw conclusions as
to (i) the validity of the structural model for M. EcoRV and
-N-MTases, (ii) the functional roles of some amino acid
residues within the conserved motifs of N-MTases, and (iii) the
catalytic mechanism of N-MTases. To our knowledge, this work
represents the first comprehensive analysis of a DNA MTase by
site-directed mutagenesis that covers positions located in all
conserved motifs as well as in the DNA recognition domain. Thus, it
complements the detailed structural information available for this
important family of enzymes.

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Fig. 1.
Alignments of M. EcoRV
with other -N-MTases. Amino acid exchanges
introduced in M. EcoRV in this work are indicated by
vertical arrows, which point to the identity of
the new amino acid. Strongly and moderately conserved residues are
boxed and shaded darkly and
lightly, respectively. The position of the putative DNA
binding domain is indicated by vertical arrows.
A, alignment of the catalytic domains of all
-N-MTases. On the basis of this structure-guided
multiple alignment, 10 conserved amino acid regions have been defined
(21). B, alignment of M. EcoRV with the seven
most closely related -N-MTases (40). The approximate
locations of conserved amino acid motifs as defined in Ref. 21 are
indicated.
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Fig. 2.
Location of mutated amino acid residues in
the structural model of M. EcoRV (40). Strongly
conserved amino acid residues are given in gray ovals, and
less conserved residues are in white ovals. The
positions of the putative AdoMet and adenine binding sites are
indicated. The structure prediction is taken from Jeltsch et
al. (40). It does not differ from that given by Malone et
al. (21) in the region of amino acid residues 1-235. -helices
and -strand are labeled A-F and 1-9.
Localizations of the secondary structure elements in primary sequence
are as follows: -helix A, 18-26; -strand 1, 42-46; -helix B,
44-48; -strand 2, 53-58; -helix C, 61-63; -strand 3, 64-70; -helix D, 78-86; -strand 4, 188-192; -helix E,
211-222; -strand 5, 224-229; -helix F, 239-248; -strand 6, 250-258; -strand 7, 270-276; -strand 8, 280-285; -strand 9, 292-299.
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EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis--
Site-directed PCR mutagenesis was
carried out on pRVMetH6, encoding the N-terminal His6-M.
EcoRV fusion protein (34, 40). In this vector, the M. EcoRV gene is controlled by a synthetic promotor-operator
sequence obtained by a combination of the T5 promotor and two
lac operators. PCR mutagenesis was performed using a
megaprimer protocol modified after Ito et al. (41). Briefly,
the method employs three PCR primers (Fig.
3): pup binds to the coding
strand upstream of the gene of M. EcoRV; plow1
binds to the lower strand downstream of the gene and removes a
SalI site 3' to the gene; and plow2 binds
downstream of plow1 but leaves the SalI site
intact. The desired mutation is introduced into the gene (together with
a marker site) by an additional upper primer (pmut) in a
PCR together with plow2. The PCR product that consists of a
part of the M. EcoRV gene and carries a SalI site 3' to the gene was hybridized to a PCR product produced using pup and plow1, which contains a
BamHI-site 5' to the gene but no SalI site 3' to
the gene. After three PCR fill in steps, a PCR using pup
and plow2 was carried out. After this procedure, only
mutated products carry a SalI site on their 3'-ends and can be cloned into the large BamHI/SalI fragment of
pRVMetH6. Usually 30-50% of the obtained clones contained the marker
site and the desired mutation. All genes were completely sequenced
after mutagenesis.

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Fig. 3.
Primers used for site-directed mutagenesis of
the M. EcoRV gene. Primer pmut introduces
a mutation into the gene of M. EcoRV (symbolized by a
triangle) along with a marker site (symbolized by a
rectangle). Note that different pmut primers
were used for the different mutations introduced. Primer
plow1 removes a SalI site downstream of the M. EcoRV gene (symbolized by ×).
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In Vivo Activity Assays--
Due to background expression,
plasmids encoding a DNA MTase are resistant to cleavage by the
corresponding restriction endonuclease. This property can be used to
assay the catalytic activity of MTases in vivo. To this end,
pRVMetHis (34, 40) or pGEXMRV (42) plasmids were grown in
Escherichia coli LK111(
) cells containing chromosomally
encoded lacIq. The transcription of the M. EcoRV
genes was not induced. Plasmid preparations from overnight cultures and
from growing cells (at 1 A600) were carried out
using DNA minipreparation kits (Qiagen) according to the instructions
of the supplier, except that in the case of growing cells 20 ml of cell
culture was used instead of 3-4 ml as used in the case of stationary
overnight cultures. Plasmid DNA (1-2 µg) was incubated with 100-200
nM R. EcoRV (corresponding to approximately 100 units) in 10 µl of Tris/HCl (pH 7.5), 10 mM
MgCl2, 50 mM NaCl for 30-60 min. Subsequently,
the samples were analyzed by agarose gel electrophoresis. Under these
conditions, an unmethylated control plasmid was completely cleaved
after 5 min, but no nonspecific DNA degradation of methylated plasmids was observed after incubation for 60 min.
Construction, Overexpression, and Purification of GST-M. EcoRV
and GST-M. EcoRV Variants--
Mutant genes were amplified from
pRVMetH6 by PCR and cloned into pGEXMRV using the BamHI and
SalI sites (42). In this vector, the M. EcoRV
gene is fused in frame to the 3'-end of the gene for the glutathione
S-transferase. The GST-M. EcoRV gene is under control of a tac promotor. Purification of the GST fusion
proteins was performed by chromatography over GSH-Sepharose and
phosphocellulose as described (42) except that cell lysis was carried
out under low salt conditions (30 mM KPi, pH
7.2.; 0.1 mM dithioerythritol; 0.01% (v/v) Lubrol; 0.5 mM EDTA, 100 mM NaCl). All mutant proteins purified in this study were obtained at similar concentrations as wild
type M. EcoRV (viz. 5-25 µM). The
specific activities of different GST-M. EcoRV preparations
(50-100 units/µg) were slightly higher than those reported for
untagged M. EcoRV (35). His6-M. EcoRV
could be purified to a 10-50-fold higher specific activity (34).
In Vitro Activity Assays--
Kinetics of restriction protection
were carried out using
-DNA (21 EcoRV sites) and
pAT153-DNA (one EcoRV site) in 50 mM NaCl, 50 mM HEPES (pH 7.5), 1 mM AdoMet at ambient
temperature as described (42). 1 unit is defined as the amount of M. EcoRV required to protect 1 µg of DNA from R. EcoRV attack in 1 h. Specific activities (units/µg of
protein) for GST fusion proteins were calculated using the molecular
mass only of the M. EcoRV part (35,000 g/mol) in order to
make the activities comparable with those measured with
His6 fusion proteins. Methylation of a 20-mer oligonucleotide d(GATCGACGATATCGTCGATC) was carried out
using [14CH3]AdoMet in 50 mM NaCl, 50 mM HEPES (pH 7.5) at ambient
temperature as described (34). To determine Km
values for DNA and AdoMet, DNA concentrations were varied between 0.2 and 4 µM at a constant AdoMet concentration of 5 µM, AdoMet concentrations were varied between 1 and 10 µM at 1 µM DNA, and the determined rates of
methylation of DNA were fit to the Michaelis-Menten model.
DNA Binding--
DNA binding was analyzed by nitrocellulose
filter experiments carried out in a dot blot apparatus (Bio-Rad) as
described (34) using a PCR product (382 base pairs) at 1 nM
and a 20-mer oligonucleotide d(GATCGACGATATCGTCGATC) (5 nM) both containing one EcoRV site as
substrates. The oligonucleotide substrate was labeled radioactively using [32P]ATP (Amersham Pharmacia Biotech) and
polynucleotide kinase (MBI Fermentas). The DNA was incubated with
different amounts of M. EcoRV variants (10-2500
nM) in 50 µl of 50 mM Tris/HCl (pH 7.5), 20 mM NaCl, 1 mM EDTA, and 200 µM
sinefungin for 30 min at ambient temperature. The nitrocellulose filter
membrane (Macherey & Nagel, Düren, Germany) was prerinsed with 50 mM Tris/HCl (pH 7.5), 20 mM NaCl for 30 min.
After membrane was transferred into the dot blot chamber, the slots
were washed twice with 100 µl of 50 mM Tris/HCl, pH 7.5, 20 mM NaCl. The samples were transferred into the wells of
the dot blot apparatus using a multiple pipette, immediately
sucked through the nitrocellulose filter membrane, and washed several
times with 100 µl of washing buffer (50 mM Tris/HCl,
pH 7.5, 20 mM NaCl). The radioactivity of the spots was analyzed using an Instant Imager (Canberra Packard), and the results were fitted to the equation describing a bimolecular
association equilibrium.
AdoMet Binding--
AdoMet binding was analyzed by filter
binding experiments using a positively charged nylon membrane
(Nytran-plus, Schleicher & Schüll) and
[carboxyl-14C]AdoMet (Amersham Pharmacia
Biotech) as described (42). M. EcoRV wild type and mutants
were incubated at
20 °C overnight with 100 µM
[carboxyl-14C]AdoMet (Amersham Pharmacia
Biotech) in storage buffer (30 mM potassium phosphate, pH
7.6, 0.1 mM dithioerythritol, 0.01% (v/v) Lubrol, 0.3 M NaCl, 0.5 mM EDTA, 77% glycerol). The nylon
membrane was prerinsed with washing buffer for at least 60 min. After
membrane was transferred into the dot blot chamber, the slots were
washed twice with washing buffer. After the incubation, the reaction mixtures containing AdoMet and M. EcoRV were diluted 10-fold
with washing buffer, transferred into the wells of the dot blot
apparatus using a multiple pipette, immediately sucked through
the nylon membrane, and washed several times with 100 µl of washing
buffer. The radioactivity of the spots was analyzed using an Instant
Imager (Canberra Packard) or PhosphorImager (Fuji). After background subtraction, the amounts of retained AdoMet were normalized to the concentrations of the enzyme preparations and compared with wild
type binding determined in the same experiment.
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RESULTS |
We have used multiple sequence alignments to identify conserved
amino acid residues in the EcoRV
adenine-N6-MTase. Two alignments were
analyzed for conserved residues, one including all
-type
N-MTases (Fig. 1A) (21) and one in which only a
subgroup of enzymes closely related to M. EcoRV are aligned (Fig. 1B) (40). On the basis of these alignments, we have
chosen 17 amino acid residues that are strongly (Lys16,
Glu37, Phe39, Asp58,
Glu104, Arg128, Asn130,
Glu178, Asp193, Tyr196,
Ser229) or moderately conserved (Asp78,
Asp211, Glu217, Glu238,
Tyr258) for this mutational study (cf. Fig. 2
for their location in the structural model). All residues were
substituted by alanine; at two positions within the conserved DPPY
motif (Asp193 and Tyr196) in addition more
conservative amino acid replacements were carried out (Asp
Asn and
Tyr
Phe). Mutations were introduced by site-directed PCR
mutagenesis. All M. EcoRV variants were tested for their
ability to methylate DNA in vivo, and most of the variants
were purified and also characterized in vitro with respect
to their catalytic properties as well as DNA binding and AdoMet binding
affinities.
Catalytic Activity of the M. EcoRV Variants in Vivo--
We have
cloned all mutants as His6 fusion proteins in pRVMetH6.
This plasmid contains one EcoRV recognition site. During
propagation of the expression plasmids coding for the individual
mutants in E. coli cells, residual expression of the MTase
variant takes place even under repressing conditions. Thus, the MTase
variant can modify the plasmid DNA in vivo, with the result
that purified plasmids are resistant against R. EcoRV
cleavage to a degree corresponding to the catalytic activity of the
MTase variant. We have carried out this in vivo activity
test in two ways by isolating plasmids either from stationary E. coli cells or from growing E. coli cells (Fig.
4, Table
I). Plasmids encoding wild type M. EcoRV are fully protected against R. EcoRV
cleavage under both experimental conditions. Generally, the degree of
methylation of EcoRV sites is lower under conditions of
active growth, because then DNA replication takes place and, in
comparison with stationary E. coli cells, higher M. EcoRV activities are required to achieve the same degree of methylation. Five of the variants fail to methylate the DNA even in
stationary E. coli cells (D58A, R128A, D193A, D193N, and
Y196A). In growing cells, many variants show a strongly reduced level of DNA methylation indicating a strongly reduced catalytic activity (
or
in Table I) (K16A, E37A, F39A, D58A, R128A, N130A, D193(A/N), Y196(A/F), S229A, Y258A).

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Fig. 4.
In vivo activity of M. EcoRV mutants. pRVMetH6 (mutant) plasmids isolated
from stationary (A) or growing E. coli cells
(B) are shown prior to (n) and after incubation
with 100 units of R. EcoRV (r). Plasmids coding
for active M. EcoRV variants are methylated and protected
from R. EcoRV cleavage. The positions of superhelical
(sc), open circle (oc), and linear
(lin) pRVMetH6 are indicated. On the basis of the degree of
protection of the plasmids coding for the M. EcoRV variants
from R. EcoRV cleavage, the catalytic activities of the
variants were assigned as: ++ (fully protected), + (>50% protected), (<50% protected), and (not protected). wt,
wild type.
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Table I
Compilation of in vivo catalytic activities of His6- and GST-M.
EcoRV mutants as indicated by the level of protection of plasmids
coding for M. EcoRV variants from R. EcoRV cleavage
All experiments were carried out at least in triplicate using different
E. coli clones expressing the M. EcoRV variants.
++, fully protected; +, >50% protected; , <50% protected; ,
not protected.
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Catalytic Activity of the M. EcoRV Variants in Vitro--
To
investigate the mutant proteins in vitro, we have
overexpressed and purified wild type M. EcoRV and 18 of the
variants (Table II). E104A and E178A that
are fully active in vivo were not purified and further
investigated. Since many mutants could not be overexpressed as
His6 fusion proteins or remained in the pellet after cell
disruption and centrifugation, we decided to express and purify all
mutants as GST fusion proteins. To this end, all mutants were also
cloned as GST fusion proteins in pGEXMRV. The catalytic activities of
these variants in stationary E. coli cells are very similar
to those of the His6 fusion proteins (data not shown). Wild
type GST-M. EcoRV and the mutants were overexpressed in
500-ml scale and purified by two chromatography steps
(glutathione-Sepharose and phosphocellulose) to >90% purity as judged
by Coomassie-stained SDS-polyacrylamide gels. The concentrations of all
preparations were between 5 and 25 µM. We have determined
the catalytic activity of wild type GST-M. EcoRV and all
mutants by
- and plasmid-DNA protection assays (Fig.
5; Table II). High catalytic activity could be detected only with wild type M. EcoRV and the D78A
mutant. The Y196F, E217A, E238A, and D244A mutants displayed a reduced activity in vitro, and all other variants were catalytically
inactive in vitro. In general, there is a very good
agreement between the in vivo and in vitro data;
most mutants with strongly reduced catalytic activities in
vivo, (
or
in growing cells, Table I), are
catalytically inactive in vitro, whereas most mutants with
good in vivo activity (+ or ++ in growing cells, Table I) also display catalytic activity in vitro. There are only two
exceptions to these rules; Y196F has a low catalytic activity in
vivo (
in Table I) but is not inactive in vitro, and
D211A is inactive in vitro but shows good activity in
vivo (+ in Table I). These differences may be caused by different
expression rates and/or different in vivo and in
vitro stabilities of these two mutants.
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Table II
Compilation of in vitro properties of GST-M. EcoRV mutants
Catalytic activities were determined using a -DNA cleavage
protection assay. DNA binding and AdoMet binding was assayed by filter
binding techniques. Errors of the DNA binding constants are estimated
to be ±30%.
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Fig. 5.
In vitro activity of GST-M.
EcoRV mutants. 0.5 µg of -DNA were incubated with
1 µl of purified M. EcoRV-GST fusion proteins (M. EcoRV, 12 µM; D78A, 13 µM;
E217A, 22 µM; S229A, 5.7 µM) diluted as
indicated in 10 µl of 50 mM NaCl, 50 mM HEPES
(pH 7.5), 1 mM AdoMet at ambient temperature for 60 min.
After the times indicated, 100 units of R. EcoRV were added
to the reaction mixtures, and MgCl2 was added to a final
concentration of 10 mM to measure the degree of methylation
of EcoRV sites. As controls, native -DNA (n)
(48,502 base pairs) and EcoRV-digested -DNA
(r) are shown.
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According to their catalytic activities, the variants can be divided
into three groups: group 1 (catalytically inactive in vivo
and in vitro), D58A, R128A, D193A, D193N, and Y196A; group 2 (catalytically inactive in vitro), K16A, E37A, F39A, N130A, D211A, S229A, and Y258A; group 3 (catalytically active in
vivo and in vitro), D78A, E104A, E178A, Y196F, E217A,
E238A, and D244A.
Kinetic Characterization of Catalytically Active M. EcoRV
Variants--
The activities of the Y196F, E217A, E238A, and D244A
variants were determined by the restriction protection assay at
concentrations of AdoMet between 1 mM and 0.3 µM (data not shown). The catalytic activities of the
variants were analyzed according to the Michaelis-Menten model to
estimate a Km value for AdoMet. According to this
analysis, the Y196F, E217A, and E238A mutants had Km values between 5 and 20 µM, similar to wild type M. EcoRV and the D78A variant (see below). In contrast, the
D244A variant shows a significantly higher Km value
for AdoMet (>250 µM), indicating a reduced affinity for
the cofactor.
In addition to performing the restriction protection assays using
macromolecular substrates, we have analyzed the in vitro activity of all mutants by determining incorporation of radioactively labeled methyl groups into a 20-mer oligonucleotide. However, apart
from wild type M. EcoRV, only the D78A variant had
sufficiently high activity to allow an unequivocal detection of
catalytic activity with this assay. We have analyzed the D78A variant
in more detail using the 20-mer substrate and determined its
Km values for DNA and AdoMet. Both kinetic constants
(Km, DNA = 0.4 ± 0.2 µM in
the presence of 5 µM AdoMet; Km,
AdoMet = 13 ± 5 µM in the presence of 1 µM 20-mer) were very similar to those measured with wild
type GST-M. EcoRV (Km, DNA = 0.3 ± 0.1 µM; Km, AdoMet = 12 ± 5 µM) and wild type His6-M.
EcoRV (Km, DNA = 0.3 µM; Km, AdoMet = 12 µM (data taken from Ref. 34)) under these conditions.
DNA Binding--
We have determined the binding constants of the
GST-M. EcoRV variants to a 20-mer oligonucleotide substrate.
Wild type GST-M. EcoRV binds to DNA with similar affinity
(Ka = 5 × 106
M
1) as His6-M. EcoRV
(Ka = 6 × 106
M
1, Ref. 34) and untagged M. EcoRV
(Ka to a 30/33-mer oligonucleotide = 2 × 107 M
1; Ref. 37). The D58A, D78A,
D193(A/N), Y196(A/F), D211A, E217A, S229A, E238A, D244A, and Y258A
variants bind to their DNA substrate with similar binding constants as
wild type M. EcoRV (Table II, Fig.
6). Some of the mutants even bind
slightly better to DNA than wild type M. EcoRV, which in
some of the cases (e.g. D58A, D193N, or D211A) might be
caused by the removal of negative charge due to the amino acid
exchange. Binding to the 20-mer could not be detected with the K16A,
E37A, F39A, R128A, and N130A. In addition, we have analyzed DNA binding
using a 382-mer substrate, because we have shown previously that this
substrate is bound better by many M. EcoRV mutants (42).
Binding to the 382-mer was detectable with all mutants that were able
to bind the 20-mer but in addition with the K16A, E37A, and F39A
variants. Only the R128A and N130A enzymes did not bind to both
substrates (Table II, data not shown). Thus, only amino acid
substitutions introduced into the putative DNA binding domain of M. EcoRV prevent DNA binding of the enzyme, whereas all
variants carrying mutations in the putative catalytic domain can bind
to DNA.

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Fig. 6.
DNA binding of the GST-M.
EcoRV variants. 5 nM 20-mer was incubated
with enzyme amounts as indicated in 50 mM Tris/HCl (pH
7.5), 20 mM NaCl, 1 mM EDTA, and 200 µM sinefungin and passed trough a nitrocellulose membrane
under vacuum. Retained radioactivity was quantified, and the data were
fitted to a bimolecular association equilibrium. All experiments were
carried out at least twice. The Ka values are given
in Table II.
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AdoMet Binding--
To determine AdoMet binding abilities of the
GST-M. EcoRV variants, a filter binding assay was employed.
In these experiments, 50 pmol of wild type GST-M. EcoRV
retain 0.5 pmol of AdoMet on the nylon membrane. At present, we cannot
explain this low retention efficiency. We do not think that most of the
protein is inactive, because with His6-M. EcoRV
the AdoMet retention efficiency was only 3-fold higher (42), although
80% of the protein in this preparation is active in DNA binding (34).
It should be noticed that here a heterophasic assay is employed to
analyze a binding equilibrium characterized by a relatively low
equilibrium binding constant (Km for AdoMet is 12 µM; Ref. 34). Under such circumstances, often not all
bound ligand molecules are retained on the filter, and stoichiometric
data cannot be obtained. Consequently, the AdoMet binding efficiencies
observed here (Fig. 7, Table II) were
only interpreted qualitatively. Four of the GST-tagged variants displayed AdoMet binding comparable with wild type GST-M.
EcoRV (D78A, R128A, D193N, and S229A). AdoMet binding was
detectable with all other variants except the K16A, E37A, F39A, and
D58A variants. It should be noted that all of these four variants carry an amino acid exchange in the putative AdoMet binding site of M. EcoRV. As shown in Fig. 7, the amount of AdoMet binding
observed in the filter binding assay is significantly lower with the
D244A variant than with D78A, Y196F, E217A, and E238A. This result is in good agreement with the results of the kinetic analyses of the
AdoMet dependence of the D78A, Y196F, E217A, E238A, and D244A variants,
because D244A has a significantly higher Km for
AdoMet than all other variants that are active in vitro.

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Fig. 7.
AdoMet binding of the GST-M. EcoRV
variants. AdoMet binding was analyzed by filter binding
experiments using a positively charged nylon membrane. The amount of
retained AdoMet per mol of enzyme is given in relation to the amount of
AdoMet bound by wild type M. EcoRV. All experiments were
carried out at least in triplicate, and error bars indicate the maximal
deviation between all experiments carried out with each mutant.
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DISCUSSION |
Implications of the Results on the Structural Model for a-type
Adenine MTases--
We have investigated 17 highly or moderately
conserved amino acid residues of the EcoRV DNA
methyltransferase by site-directed mutagenesis. Most variants were
purified and analyzed in vitro with respect to their
catalytic activities as well as DNA binding and AdoMet binding
affinities. The mutants showing a reduced catalytic activity can be
divided into three groups: group A (AdoMet binding-deficient), K16A,
E37A, F39A, and D58A; group D (DNA binding-deficient), R128A and N130A;
group C (detectable AdoMet and DNA binding, strongly reduced
catalytic efficiency), D193(A/N), Y196A, S229A, D211A, and
Y258A.
All mutants of groups A and D are catalytically inactive or show a
severely reduced catalytic efficiency. The locations of the variants
from groups A, D, and C in the structural model are shown in Fig.
8. Obviously, excellent agreement exists
between the structural model and the biochemical properties of the
mutants, because all variants of group A (no AdoMet binding) are
located at the putative AdoMet binding site, all variants of group D
(no DNA binding) in the putative DNA binding domain, and all variants of group C (only catalysis affected) at the putative binding site of a
flipped target base. These results support the structural model of M. EcoRV. In the following paragraphs, the biochemical properties of the variants will be discussed in more detail.

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Fig. 8.
Location of groups of variants in the
structural model of M. EcoRV (cf. Fig.
2). A, location of residues exchanged in variants
unable to bind AdoMet (group A: Lys16, Glu37,
Phe39, and Asp58). B, location of
residues exchanged in variants unable to bind to DNA (group D:
Arg128 and Asn130). C, location of
residues exchanged in variants that bind to AdoMet and DNA but show a
strongly reduced catalytic activity (group C: Asp193,
Tyr196, Asp211, Ser229,
Trp231, and Tyr258).
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AdoMet Binding--
Four of the mutants investigated are unable to
bind AdoMet in vitro (K16A, E37A, F39A, D58A). All of these
mutants are catalytically inactive in vitro and show a
strongly reduced catalytic efficiency in vivo. These
variants are mutated at the following amino acid residues that are
implicated in AdoMet binding of M. EcoRV. Lys16
(motif X) is analogous to Thr23 in M. TaqI and
Asn304 in M. HhaI, both of which contact one of
the carboxyl oxygens of the AdoMet. Glu37 (motif I)
corresponds to Glu45 in M. TaqI and
Asp16 in M. HhaI, which contact the peptide
backbone within motif I (FXGXG), thereby
stereochemically constraining this region. In addition, M. TaqI-Glu45 contacts the amino group of the
methionine moiety of the AdoMet. Phe39 (motif I) is
equivalent to Phe18 in M. HhaI, which forms a
hydrophobic contact to the adenine ring of the AdoMet (the Phe in the
motif I absent in M. TaqI). Asp58 (motif II)
corresponds to Glu40 in M. HhaI and
Glu71 in TaqI, which hydrogen bond to 2'- and
3'-oxygens of the ribose of the AdoMet.
Our results suggest that all of these contacts to the cofactor observed
in the x-ray structure analyses of several methyltransferases (M. HhaI (7), catechol-O-methyltransferase (43), M. TaqI (28), HaeIII DNA methyltransferase (15),
VP39 vaccinia protein RNA methyltransferase (44), CheR (45), and
PvuII DNA methyltransferase (29)) are very important for
binding and positioning the cofactor. The data obtained with
Glu37 and Phe39 in motif I
(FXGXG) complement other mutational studies
concerned with this region, because exchanging the first glycine of the FXGXG by other amino acid residues prevents
AdoMet binding by EcoKI and EcoP15I (23, 24).
Interestingly, K16A, E37A, and F39A also show a reduced DNA binding
capacity. This finding is in good agreement with data obtained for wild
type M. EcoRV showing that DNA binding is about 10-fold
stronger in the presence than in the absence of cofactor (37) similarly
as described for other DNA MTases (EcoRI DNA methyltransferase (46), MspI DNA methyltransferase (47), and HaeIII DNA methyltransferase (48)). It has been suggested
that an allosteric effect connecting conformational changes at the DNA
and AdoMet binding sites is responsible for this effect (49). In
addition to the residues mentioned above, the D244A mutant shows a
significantly reduced affinity for AdoMet. If the structural model
(Fig. 2) is correct in this region, this effect could only be mediated
allosterically because
-helix F is far away from the AdoMet binding
site in M. TaqI and M. HhaI.
The properties of the D78A variant are very different from what one
would have expected on the basis of the structural model for M. EcoRV, because according to the model this residue should correspond to Asp89 in M. TaqI and
Asp60 in M. HhaI that contact the adenine
N6 of the AdoMet. However, D78A does not show any evidence
for a reduced affinity toward AdoMet or a reduced catalytic efficiency; it has high catalytic activity both in vivo and in
vitro, it has a similar Km value for AdoMet as
wild type M. EcoRV, and it binds as much AdoMet as wild type
M. EcoRV in the AdoMet binding assay. We conclude that
either the structural model for M. EcoRV is wrong in this
detail or this contact is of minor importance for AdoMet binding.
Therefore, we have looked for alternative candidate residues to fulfill
the function of Asp89 in M. TaqI.
Glu104 is the best conserved acidic amino acid residue in
putative DNA binding domain, and Glu178 is highly conserved
and positioned such that if M. EcoRV had similar structure
as observed for PvuII DNA methyltransferase it would be
located at the edge of
-helix D. However, exchanging these residues
to alanine did not provide evidence for an important role of them.
Since there are several other alternative candidate residues,
e.g. Asp74, Asp111,
Asp187, and Asp188, we think that this question
is still unresolved.
DNA Binding--
The DNA binding domain of M. EcoRV was
predicted to comprise amino acid residues 90-180 (40). In this study,
we have investigated two residues within this region
(Arg128 and Asn130). R128A and N130A both are
not able to bind DNA but can bind the cofactor AdoMet. These are the
only residues in this study that are unable to bind to DNA. This
finding is comparable with similar results of a random mutagenesis
study that identified 13 M. EcoRV single mutants that are
catalytically inactive in vivo and in vitro. Five
of these variants do not bind to DNA, four of which carry a mutation in
the putative DNA binding domain (42).
Catalytic Mechanism--
There is evidence that N-MTases flip out
their target adenine prior to methyl group transfer (31-34). Thus, the
active site of the enzyme must form a binding site for the flipped out
adenine residue, providing a hydrophobic pocket and hydrogen bond
partners for the adenine as well as catalytic residues. It should be
noted that variants that are not able to flip out the target base are not expected to have a lower affinity toward DNA, because nonspecific binding of M. EcoRV to DNA is almost as strong as specific
binding (34). Two of the most conserved mutants investigated here
(D193N, S229A) are catalytically inactive but bind to DNA and AdoMet
with similar affinities as wild type M. EcoRV.
Ser229 and Asp193 consequently are prime
candidate residues to be involved in catalysis. This conclusion is not
surprising for Asp193, because the aspartic acid of the
DPPY motif (motif IV) almost certainly is part of the catalytic site of
N-MTases (30, 31). Our results with the D193N and D193A
variants are in agreement with mutational data obtained with
EcoKI, BcgI, and E. coli Dam (22, 23,
25); a DPPY
NPPY exchange in E. coli Dam (an
-N-MTase) abolishes catalytic activity but leaves DNA
binding of the enzyme intact. Interestingly, AdoMet binding could not be demonstrated with this variant (22). In EcoKI, an NPPF
DPPF exchange results in a catalytically inactive enzyme that still
binds the cofactor (23) and variants of BcgI in which the
NPPY Asn was exchanged by Ala, Asp, or Gln are catalytically inactive (25).
Ser229, which is highly conserved in
-N-MTases is located in motif VI and corresponds to
Glu119 in M. HhaI. This residue is involved in
acid base catalysis in M. HhaI (13). It should be noted that
the reaction catalyzed by M. EcoRV, methylation of an
adenine N6, requires the abstraction of one proton from the
target base. In contrast to
-N-MTases in
-type enzymes
(mostly cytosine-N4-MTases), an acidic
residue is located at a corresponding position in motif VI
(Asp96 in PvuII DNA methyltransferase) (29).
However, motif IV has a SPPF sequence in these enzymes. Thus, it
appears as if the active site of
-type enzymes
(DPPY ... S) is analogous to the
SPPF ... D arrangement found in
-type
enzymes. Hence, Ser229 possibly together with
Asp193 could be involved in a proton relay system
(adenine ... Ser229 ... Asp193)
similarly as suggested recently for the
-type N-MTase
PvuII methyltransferase (29). Alternatively,
Ser229 may form a hydrogen bond to the flipped adenine.
In addition, Asp211 is important for the catalytic activity
of M. EcoRV, because the D211A variant is inactive in
vitro, although this variant shows considerable activity in
vivo. This result is in agreement with the structural model,
according to which this residue is located in motif V, that
participates in creating the adenine binding pocket. Asp211
is located at a position corresponding to Phe146 in M. TaqI that forms a hydrophobic contact to the AdoMet.
However, the Phe146-AdoMet interaction is characteristic
for
-type N-MTases, and our data do not suggest
involvement of the corresponding residue in M. EcoRV
(Asp211) in AdoMet binding.
Adenine Binding Site--
In addition to Asp193,
Tyr196 is the second conserved amino acid residue in motif
IV investigated in this study. In M. TaqI, a hydrogen bond
is formed between the hydroxyl group of this tyrosine and the conserved
asparagine constraining the conformation of the NPPY motif (motif IV).
Perhaps, in M. EcoRV Tyr196 serves to position
Asp193 by having a buttressing role in catalysis. In
agreement with this observation, we show here that the hydroxyl group
of Tyr196 contributes to catalysis but is much less
important than the carboxylate group of Asp193. However,
our results obtained with the Y196A variant demonstrate that an
aromatic residue is essential at this position, presumably because it
is involved in binding the flipped adenine. These findings are in
agreement with data obtained with EcoKI where the Phe
residue of the NPPF motif was replaced by Tyr and Trp and the resulting NPPY and NPPW variants still had catalytic activity (1/4 of wild type in the case of NPPY, much less in the case of NPPW) (23). In
BcgI, an NPPY
NPPA exchange abolishes catalytic activity but does not severely affect DNA binding and AdoMet binding. Similarly as found here, NPPY
NPPF or NPPW variants of BcgI retain
catalytic activity (25). In contrast, a DPPY
DPPW exchange in
EcoP15I inactivates this enzyme (24).
A second candidate residue to contribute to the adenine binding pocket
is Trp231, which has been identified in a random
mutagenesis approach (42). A W231R mutant binds to DNA and AdoMet
similar to wild type M. EcoRV but is catalytically inactive
in vivo and in vitro. According to the structure
prediction for M. EcoRV, Trp231 is located at a
position corresponding to Val121 in M. HhaI
(40). This residue forms a hydrophobic contact to the flipped cytosine
(12), suggesting that Trp231 in M. EcoRV may
have a similar function in binding the flipped adenine.
Finally, Tyr258 (motif VIII) could participate in binding
the flipped out adenine. This residue could provide an aromatic ring and/or hydroxyl group to interact with the adenine base. It corresponds to Phe196 in M. TaqI, which has been suggested
to be involved in hydrophobic contacts to the flipped adenine ring (21,
31). Our data demonstrate that Tyr258 indeed is an
important residue for catalysis, because the Y258A variant is
catalytically almost inactive but binds to DNA and AdoMet, albeit with
reduced affinity for the cofactor.
Conclusions--
In this site-directed mutagenesis study covering
17 positions of M. EcoRV, we have provided evidence that a
model for the structure of
-type N-MTases is largely
correct. We could demonstrate the importance of amino acid residues in
motifs X, I, and II in AdoMet binding. In addition, residues in motifs
IV, V, VI, and VIII are important for catalysis and/or might contribute
to the binding site for a flipped adenine. These data considerably
extend our knowledge on DNA MTases, because so far results of
site-directed mutagenesis studies were only published for some residues
located in motifs I and IV. Together with more structural information, they will help us to understand better structure-function relationships and mechanism of DNA N-MTases.
We are grateful to Drs. X. Cheng, B. A. Connolly, N. O. Reich, W. Saenger, and E. Weinhold for discussions
and/or communication of results prior to publication. Thanks are due to
Dr. A. Pingoud for insightful comments and support as well as to H. Büngen for excellent technical assistance. We acknowledge the
generous gift of sinefungin by Dr. W. Guschlbauer.