From the
The bacteriophage T4 dam gene, encoding the Dam DNA
[N
The K
DNA methyltransferases (MTases)
We have constructed a plasmid that overexpresses the T4 Dam
DNA MTase in a regulatable fashion. This was accomplished by using an
expression vector, pJW2(13) , containing the strong phage
T4 Dam, at a molecular weight
of 30.4 kDa, is one of the smallest MTases that exists as monomer in
solution, such as M.EcoDam (32 kDa)(25) ,
M.EcoRI (39 kDa)(8) , and M.HhaI (37
kDa)(21) . Most characterized MTases exist as monomers in
solution (26), although recently it has been reported that
M.RsrI shows partial dimerization(24) ; and two MTases
from Streptococcus pneumoniae appear to exist as
dimers(27) . In the case of M.MspI, the enzyme appears
to dimerize at high protein concentrations, which may reflect a
tendency to aggregate rather than having functional
significance(18) . The sensitivity of T4 Dam to N-ethylmaleimide indicates that one or more cysteine residues
is important for activity. Similar requirements have been observed for
the adenine MTases, M.Eco Dam and
M.EcoRI(25, 28) .
T4 Dam is also similar to
bacterial Type II DNA MTases with respect to kinetic parameters, except
that it has lower K
The
ability of T4 Dam to methylate noncanonical sites in vitro was
demonstrated by conferring protection against cleavage by
methylation-sensitive restriction endonucleases that recognize
sequences partially overlapping GATC. These results are consistent with
studies on in vivo methylation by T4 Dam(3) . It was
previously shown that phage T2 Dam (which differs from T4 Dam in three
amino acid residues) can methylate sites other than GATC in
vitro(29) . The bacterial MTases,
M.EcoRI(30) , M.EcoRV (31) and
M.EcoDam(32) , can methylate noncanonical sites to a
limited extent, but only under special conditions in vitro.
The availability of highly purified T4 Dam will facilitate
crystallization trials and further analyses of the enzyme, particularly
with respect to interaction with its target site(s) on DNA.
-adenine]methyltransferase (MTase), has
been subcloned into the plasmid expression vector, pJW2. In this
construct, designated pINT4dam, transcription is from the
regulatable phage
p
and p
promoters, arranged in tandem. A two-step purification scheme
using DEAE-cellulose and phosphocellulose columns in series, followed
by hydroxyapatite chromatography, was developed to purify the enzyme to
near homogeneity. The yield of purified protein was 2 mg/g of cell
paste. The MTase has an s
of 3.0 S and a Stokes radius of 23 Å and exists in
solution as a monomer.
for the
methyl donor, S-adenosylmethionine, is 0.1
10
M, and the K
for substrate nonglucosylated, unmethylated T4 gt
dam
DNA is 1.1
10
M. The products of DNA
methylation, S-adenosyl-L-homocysteine and methylated
DNA, are competitive inhibitors of the reaction; K
values of 2.4
10
M and
4.6
10
M, respectively, were
observed. T4 Dam methylates the palindromic tetranucleotide, GATC,
designated the canonical sequence. However, at high MTase:DNA ratios,
T4 Dam can methylate some noncanonical sequences belonging to GAY
(where Y represents cytosine or thymine).
(
)are
ubiquitous in living cells and present an interesting example of
sequence-specific DNA-protein interaction. DNA MTases recognize
specific sequences in DNA and transfer methyl groups from the methyl
donor, AdoMet, to adenine or cytosine residues in the recognition
sequence. Three kinds of methylation product are known, N
-methyladenine, N
-methylcytosine, and 5-methylcytosine. Most of
the known DNA MTases are part of restriction-modification systems, but
there are MTases that exist in the cell without a corresponding cognate
endonuclease(1) . Bacteriophage T4 encodes a DNA
[N
-adenine]MTase (T4 Dam) that methylates
adenine in the sequence GATC in cytosine, 5-methylcytosine, or
5-hydroxymethylcytosine-containing DNA(2) . At a much lower
efficiency, T4 Dam also recognizes some noncanonical sites in sequences
derived from GAY (where Y represents cytosine or thymine)(3) .
The dam gene, which encodes T4 Dam, was cloned, and its
nucleotide sequence was determined(4, 5) . We have been
especially interested in defining the region(s) of T4 Dam responsible
for sequence-specific DNA recognition as well as the functional roles
of conserved motifs present in all DNA
[N
-adenine]MTases(6) . In this paper,
we describe the overexpression, purification, and characterization of
kinetic parameters for T4 Dam.
Materials
[methyl-H]AdoMet
was purchased from DuPont NEN. Unlabeled AdoMet (Sigma) was purified by
high pressure liquid chromatography on a C18 reversed-phase column. The
concentration was calculated using a molar extinction coefficient at
256 nm of 14,700 (in acid)(7) . AdoHcy, DTT, and N-ethylmaleimide (Sigma); DE81 filters, phosphocellulose (P11)
and DEAE-cellulose (DE-52) (Whatman), hydroxyapatite (Bio-Rad),
Sephacryl S-100 (Pharmacia Biotech Inc.), restriction enzymes and DNA
ligase (New England Biolabs), and Nonidet P40 (BDH Chemicals) were
purchased from the companies in parentheses.
Bacterial Growth
Escherichia coli GM 2971
(Fmrr
hsd S20
r
m
- ara14 proA2 lacY1 galK2 rspL20 (str
) xyl5 mtl1 supE44 dam13::Tn9 Cm
) was from Dr. M. G. Marinus,
Department of Pharmacology, University of Massachusetts Medical School,
Worcester. Plasmid-containing cells were grown in LB-ampicillin broth (8) at 30 °C to an A
= 0.8-1.0. The temperature was raised to 42 °C, and incubation
continued for 3 h. The cells were collected by low speed centrifugation
and stored frozen at -20 °C.
DNA MTase Assay
Methyl transfer assays were
carried out by a DE81 ion exchange filter assay(9) . Assay
mixtures (50 µl) contained 20 mM Tris-HCl, pH 8.0, 1
mM EDTA, 1 mM DTT, and 200 µg/ml bovine serum
albumin. Substrate DNA and [methyl-H]
AdoMet concentrations were as noted in each experiment.
Purification of T4 Dam
Frozen cells (18 g) were
suspended in 60 ml of PEM buffer (20 mM KPO, 1
mM EDTA, 7 mM 2-mercaptoethanol) with 0.1 mM phenylmethylsulfonyl fluoride, 0.1% of Nonidet P-40, and 0.4 M NaCl. Lysozyme was added to a concentration of 500 µg/ml and,
after a 1-h incubation at 4 °C, the cells were disrupted by
sonication. Insoluble debris was removed by centrifugation at 100,000
g for 1 h. The supernatant was diluted 2-fold in PEM
buffer and passed through a DEAE-cellulose (DE52) column connected to a
1.5
10-cm P11 phosphocellulose column (equilibrated in buffer
PEM with 0.2 M NaCl). Proteins bound to the P11
phosphocellulose were eluted in a 200-ml, 200-800 mM NaCl gradient in PEM buffer. DNA MTase activity was monitored as
described above; appropriate fractions were pooled, diluted 2-fold with
buffer PEM, and applied to a 1
4-cm hydroxyapatite column
(equilibrated in PEM buffer with 0.2 M NaCl). Proteins bound
to the matrix were eluted with a 50-ml gradient of 20-500 mM potassium phosphate with 1 mM EDTA, 7 mM 2-mercaptoethanol, and 0.2 M NaCl, pH 7.4. Active
fractions were concentrated on a 1.0-ml phosphocellulose column. The
enzyme was dialyzed against PEM with 0.1 M NaCl and 50%
glycerol and stored at -20 °C. Protein was determined by the
method of Bradford(10) .
Gel Filtration
Purified T4 Dam MTase was subjected
to gel filtration on Sephacryl S-100 in PEM buffer with 0.1 M NaCl. The void volume was determined with blue dextran. Marker
proteins bovine serum albumin (R = 37.0 Å),
ovalbumin (R
= 27.6Å), bovine carbonic
anhydrase (R
= 24.3Å), and lysozyme
(R
= 20.6Å) were used to calibrate the
column.
Glycerol Gradient Sedimentation
Centrifugation was
performed in 12.5-ml glycerol gradients (10-30%, w/v) containing
PEM buffer with 0.1 M NaCl and 50-100 µg of T4 Dam
MTase. Centrifugation was for 48 h at 40,000 rpm at 4 °C in a
Beckman SW41 rotor. Sedimentation coefficients were determined using
bovine serum albumin (4.31 S), ovalbumin (3.55 S), bovine carbonic
anhydrase (2.85 S), and bovine heart cytochrome c (1.83 S) as
standards.
Steady State Studies
Apparent K (DNA), K
(AdoMet), and k
values were determined by monitoring the
initial velocities of [
H]methyl transfer from
labeled AdoMet to nonglucosylated, unmethylated T4 gt
dam
DNA. Kinetic
parameters were obtained using the EnzymeKinetics program from Trinity
Software.
Gel Electrophoresis
SDS-PAGE was carried out
according to Laemmli(11) , and agarose gel electrophoresis was
performed in Tris acetate buffer(12) .
Construction of a T4 Dam-overproducing Plasmid
Fig. 1shows the overall scheme for cloning the T4 dam gene into the expression vector, pJW2(13) . As a source of
this gene, we used the plasmid pSSH10, which contains T4 dam on a 1.6-kilobase fragment between the HindIII and BamHI sites in pBR322(14) . We eliminated the NdeI site in the T4 dam fragment and created a SmaI site in its place. The SmaI-BamHI
fragment was cloned into pGC7 (a derivative of pGC1 (15) with a
polylinker). This construct was then used to create an NdeI
site at the ATG start of the gene (by oligonucleotide site-directed
mutagenesis). Finally, the NdeI-BamHI fragment was
excised and cloned into the vector pJW2. The resulting plasmid,
pINT4dam, is shown in Fig. 1.
Figure 1:
Construction of plasmid pINT4dam for overexpression of T4 Dam. Expression of the T4dam gene is under control of the tandemly arranged phage
promoters, p
and p
. bla,
-lactamase gene; ori, colE1 origin of
replication; cI
, phage
gene encoding a
thermolabile repressor; T7 RBS, translation initiation region of the T7
phage gene 10; and fdtt, fd transcription
terminator.
T4 Dam MTase induction
and overproduction is illustrated in Fig. 2. SDS-PAGE analysis
revealed a strongly enhanced band at the position expected for T4 Dam
(molecular mass = 30.4 kDa) (Fig. 2). The maximum amount
of T4 Dam protein was observed after 4 h and remained constant at this
level for at least another 4 h (Fig. 3). In contrast, the amount
of soluble T4 Dam MTase activity reached a maximum at 3 h and then
declined slowly thereafter.
Figure 2:
Overexpression of T4 Dam. E. coli cells carrying a T4 Dam-overproducing plasmid were grown as
described under ``Experimental Procedures.'' An equivalent
number of cells were removed at various time intervals and lysed, and
proteins were analyzed by SDS-PAGE (12.5%). The time (hrs. after
induction) after induction is shown above each lane. Lanes denoted by MW each contained
molecular mass protein standards (given in kDa); the lane denoted as T4 Dam contained purified
enzyme.
Figure 3:
Kinetics of T4 Dam overproduction. E.
coli cells growing at 30 °C were shifted up to 42 °C. At
various time intervals, aliquots were removed to determine optical
density at 600 nm and MTase activity. Cells were lysed by sonication,
and insoluble debris was removed by centrifugation. The supernatants
were assayed for MTase activity as described under ``Experimental
Procedures.'' Circles, MTase activity; squares, A.
Purification of T4 Dam
A two-step chromatography
procedure (see ``Experimental Procedures'') provided an
excellent means to obtain highly purified T4 Dam MTase (Fig. 4).
SDS-PAGE analysis of fractions from the hydroxyapatite column revealed
a single 30-kDa band corresponding to the DNA MTase activity. A summary
of purification from 18 g of E. coli cell paste is presented
in .
Figure 4:
Purification of T4 Dam. Samples collected
at each purification step were analyzed by 15% SDS-PAGE as described
under ``Experimental Procedures.'' Lane1,
crude extract; lane2, supernatant after
centrifugation at 100,000 g; lane3,
pool fraction of P11 phosphocellulose column; lane4,
pool fractions of hydroxyapatite column; lane5,
pellet after high speed centrifugation of sonicated cells; lanesMW, protein standards (in
kDa).
Molecular Weight Determination
Purified T4 Dam
MTase was subjected to glycerol gradient centrifugation and gel
filtration under native conditions. It had an s of 3.0 S and a Stokes
radius of 23 Å. We calculated a molecular weight of 30,690, using
a partial specific volume of 0.7445(16, 17) . The
apparent molecular mass of T4 Dam was 30 kDa, as determined by
SDS-PAGE. This is in agreement with the value of 30.4 kDa from the
deduced amino acid sequence. These results suggest that T4 Dam exists
as a monomer in solution.
Requirements for T4 Dam Methylation
T4 Dam, like
other DNA MTases, does not require Mg, and it is
fully active in the presence of 10 mM EDTA. T4 Dam has a broad
pH optimum (7.0-8.5) and is inhibited at ionic strengths greater
than 0.2 M. At 30 °C with low enzyme:DNA ratios, T4 Dam
maintained a constant transfer rate of labeled methyl groups from
AdoMet to DNA over a period of at least 3 h (data not shown). The
enzyme has one or two essential cysteine residues, since activity was
abolished by N-ethylmaleimide.
Steady State Kinetics
Steady state kinetic
parameters of T4 Dam methylation were studied using
T4gtdam
DNA, which is
a natural substrate for the enzyme (Fig. 5). Initial rates of
methylation were first-order with respect to enzyme concentrations up
to 5 nM, and Michaelis-Menten kinetics were obeyed with
respect to both AdoMet and DNA. The K
values were 0.1 µM for AdoMet and 1.1 pM for T4 DNA. The V
was calculated to be 270
nmol
min
mg
enzyme, and
the k
was calculated to be 0.14
s
. The k
/K
for T4 gt
dam
DNA was 0.13
10
M
s
, and for AdoMet it was 1.4
10
M
s
. Methylated
DNA was a competitive inhibitor of MTase activity with an inhibition
constant, K
, of 4.6 pM (Fig. 5B). As with other DNA MTases, product
(AdoHcy) generated by the methylation reaction was a competitive
inhibitor (with respect to AdoMet); the K
was 2.4 µM (Fig. 5A).
Figure 5:
Steady
state kinetics of T4 Dam MTase. The apparent steady state kinetic
parameters were obtained at 37 °C with 0.36-10.0 pM T4 gtdam
DNA,
0.05-1.32 µM AdoMet in 100 mM Tris, pH 8.0,
1 mM EDTA, 200 µg/ml bovine serum albumin, 1 mM DTT, 0.3-0.6 nM MTase, and varying concentrations
of AdoHcy (A) or methylated DNA (B). Methylated DNA
was prepared by extensive methylation of
T4gt
dam
DNA by T4 Dam
MTase using unlabeled AdoMet; after deproteinization, the DNA no longer
served as methyl group acceptor with T4 Dam MTase and
[methyl-
H]AdoMet.
In Vitro Methylation of Noncanonical Sites
Phage
and pUC18 DNAs were methylated by T4 Dam in vitro and
then analyzed by digestion with a set of methylation-sensitive
restriction nucleases, viz.ClaI, EcoRV, HinfI, FokI, and HphI, all of which
recognize a sequence overlapping GATC, the canonical T4 Dam-methylation
site. Previous studies had shown that T4 Dam can methylate noncanonical
sites in vivo, provided that high concentrations of enzyme
were present(3) . As seen in Fig. 6, in vitro T4
Dam methylation protected against cleavage by ClaI (ATCGAT), EcoRV (GATATC), HinfI (GATT, GACT), and FokI
(GATG) but not against HphI (GAA and GAG sites). These results
demonstrate that in vitro T4 Dam can methylate some subset of
the noncanonical sequence, GAY (where Y represents cytosine or
thymine).
Figure 6:
Methylation of noncanonical sites in
heterologous DNAs (phage (A) and plasmid pUC18 (B). Methylation was performed under standard assay conditions
using unlabeled AdoMet as methyl donor. Following incubation (for 1 h)
with increasing concentrations of T4 Dam (from 0 to 1 µg/µg of
substrate DNA, lanes 1-8 (A); from 2 pg to 1
µg/µg of substrate DNA, lanes 1-7 (B)),
the DNA was extracted with phenol/chloroform and precipitated in
ethanol. Methylated DNAs were resuspended in appropriate buffers,
digested with endonuclease ClaI (ATCGAT), EcoRV
(GATATC), FokI (GATG), or HinfI (GATT, GACT), and
then subjected to agarose gel electrophoresis. Undigested phage
DNA and HaeIII-digested phage
X174 DNA were run as
molecular weight markers.
promoters, p
and p
, arranged
in tandem, to drive transcription following heat inactivation of the
thermolabile
cI
repressor; in addition, the ribosome
binding site of the phage T7 gene 10 was present to enhance
translation initiation. Following induction, T4 Dam constituted more
than 10% of total cellular protein, but a major portion of the enzyme
sedimented as inclusion bodies along with insoluble cellular debris.
This situation is similar to that observed with several other MTases,
including M.MspI(18) , M.HhaI(19) , and
M.EcoRV(20) ; in contrast, a number of MTases have been
overproduced in soluble form, such as M.HhaI(21) ,
M.HhaII(22) , M.EcoRI(23) , and
M.RsrI (24). The formation of inclusion bodies is often
dependent upon the overexpression system and growth conditions. The
fraction of soluble T4 Dam reached a maximum by 3 h postinduction, and
then slowly declined. We describe a two-step purification procedure,
which yielded T4 Dam in apparently homogeneous form. With this method,
we were able to recover active T4 Dam MTase corresponding to about 3%
of the total cellular soluble protein.
values for substrates
and a higher catalytic rate constant. The relatively high specificity
constant of T4 Dam (k
/K
= 1.4
10
M
s
) also indicates that it is a more efficient
enzyme than the other MTases studied so far. Methylated DNA is a
competitive inhibitor of MTase activity (K
= 4.6 pM), indicating that T4 Dam exhibits
(as for other MTases) stronger (almost 4-fold) affinity for
unmethylated DNA compared with methylated DNA. The other product of
methylation, AdoHcy, also acts as a competitive inhibitor of AdoMet (K
= 2.4 µM); the
ratio K
/K
=
24 indicates that T4 Dam has a higher affinity for AdoMet.
Table: Purification of T4 Dam MTase
, potassium
phosphate; PAGE, polyacrylamide gel electrophoresis.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.